Adenine Blood Preservation: Critical Reviews in Clinical Laboratory Sciences February 1981

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Adenine Blood Preservation
Article in Critical Reviews in Clinical Laboratory Sciences · February 1981

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January 1981 173
ADENINE IN BLOOD PRESERVATION

Authors: Carl C . Peck
Division of Clinical Pharmacology
Uniformed Services University of the
Health Sciences
Bethesda, Maryland
Gerald L. Moore
Robert B. Bolin
Division of Blood Research
Letterman Army Institute of Research
Presidio of San Francisco, California
Referee: R . Ben Dawson
Department o f Pathology
University o f Maryland School of Medicine and Hospital
Baltimore, Maryland
I . INTRODUCTION
U.S.Food and Drug Administration (FDA) approval of the anticoagulant-preserv-

ative, Anticoagulant Citrate-Phosphate-Dextrose-Adeninesolution (CPDA-1) in Au-
gust 1978 marked the first major change in the available blood preservation systems
in 11 years.’ Introduction of CPDA-1 for clinical use in the U.S. came almost 2 decades
after Nakao et al.2-5first reported that adenine and inosine promote red cell adenosine
triphosphate (ATP) synthesis. Simon and colleagues translated this observation into
potential blood preservation technology by demonstrating that small amounts of ad-
enine (0.5 to 1.O mM, whole blood concentration) added t o Anticoagulant-Citrate-
Dextrose solution (ACD, Table 1) preservation media resulted in improved posttrans-
fusion “viability” following storage periods of up to 46 days.6 The reports of Simon
and Sugita’ and Dern, Brewer, and Wiorkowski” confirmed the suspected relation-
ship between erythrocyte A T P content and posttransfusion red cell survival which fur-
ther alerted investigators in blood preservation to develop strategies, including adenine
supplementation, for maintaining red cell A T P during storage. Clinical evaluation and
use of ACD-A (Table 1) blood preservative commenced in Sweden in 1964 and within
a few years in West Germany, but concern regarding the potential nephrotoxic effects
of the adenine metabolite, 2,s-dihydroxyadenine (DOA), slowed progress towards clin-
ical use in the U.S.& In 1974, an American multi-institutional cooperative research
effort was launched which aimed at collecting the necessary scientific and clinical evi-
dence for FDA consideration to approve CPDA-l for clinical use. More complete sum-
maries of the fascinating history of adenine in experimental and clinical blood preser-
vation were published in the July-August 1977 issue of T r a n s f ~ s i o dand
. ~ elsewhere.”
Timely reviews of the pharmacology. toxicology, biochemistry, and metabolism of
adenine in mammalian systems as well as the first report of the American cooperative
CPDA-I evaluations also appeared in the Transfusionissue. ’
The goal of the present review is to appraise critically our state of knowledge of the
use of adenine in blood preservation and to interpret the certainties and uncertainties
resulting therefrom in the light of trends in blood preservation research and blood
banking practice.
11. BIOCHEMISTRY
A. Transport of Adenine into Red Cells

The kinetics of purine transport into human red blood cells has not been examined
174 CRC Critical Reviews in Clinical Laboratory Sciences
Table 1
ANTICOAGULANT-PRESERVATIVECONTENTS
Designation Contents
ACD Anticoagulant-Citrate-DextroseSolution
ACD-A ACD with 0.5 m M adenine
CPD Anticoagulant Citrate-Phosphate-DextroseSolution
CPD-A CPD with 0.25 m M adenine
CPD-2A CPD with 0.50 m M adenine
CPDA- I CPD with 25% extra dextrose + 0.25 m M adenine
CPDA-2 CPD with 75% extra dextrose + 0.50 rnM adenine
CPDA-3 CPD with 100% extra dextrose + 0.50 m M adenine
a
mMadenine in final whole blood concentration.
as quantitatively and mechanistically as that of carbohydrate transport, but several

qualitative studies have shown avid incorporation of purines into red cells. Whittam"
measured the 30-min uptake of adenine into red cells which had been stored in ACD
for 28 days after washing the cells with isotonic saline and suspending them in phos-
phate buffer. He showed that at 37°C about 53% of the adenine was "taken up" by
.
the red blood cells when no carbohydrate substrate was present, but when the incuba-
tion medium also contained glucose o r inosine, the uptake was greater than 95% by
30 min. La~sen''-~'' studied hypoxanthine transport in human erythrocytes and from
kinetic data postulated a complex two-component transport mechanism: at low con-
centrations a saturable carrier process was operative, while at higher hypoxanthine
concentrations a nonsaturable diffusion process dominated. This kinetic pattern is sim-
ilar t o the parallel Michaelis-Menten (low concentration) and first-order (high concen-
tration) transport of serotonin into platelets observed by Stahl and Meltzer." Lassen
speculated that since adenine and hypoxanthine inhibit the uptake of each other, they
share a common purine transport system.
Several investigation^^^.'*-^^ of adenine uptake and utilization by human red cells
have been published using in vitro systems which mimic blood bank storage conditions.
In 1965, Sugita and Simonz5added adenine to ACD whole blood (0.6 mMfinal con-
centration) and after 6 weeks of storage observed a 50% loss of the adenine. The fall
of blood adenine was rapid for the first 3 weeks, then more gradual for the remaining
3 weeks. The reported values for blood adenine were apparently for whole blood,
which suggests incorporation into nucleotides, but does not define rates of adenine
transport into the cells. However, the authors stated that the ratio of intracellular to
extracellular adenine concentration was maintained at 1.8 throughout the storage pe-
riod. This would imply that an equilibrium between plasma and erythrocytic adenine
pools was maintained throughout the 6 weeks of storage such that adenine metabolized
inside the red cell is reflected by a proportionate fall in the plasma adenine concentra-
tion. Moore and studied the uptake of adenine by human red blood
cells in units of blood which were drawn in citrate-phosphate-dextrose-adenine (CPD-
A). They** demonstrated (Figure 1) that when 17.5 mg of adenine is added to 500 m l
of CPD whole blood, there is a rapid initial equilibration of adenine between plasma
and red cells. This initial partitioning of adenine between plasma and red cells is tem-
perature dependent, coming to equilibrium in about 20 min at 0°C and equilibrating
in less than 2 min at 20°C. When the whole blood is then stored at 4°C for 42 days,
the plasma adenine concentration gradually falls to between 10 and 20% of the initial
equilibrium value, implying gradual intraerythrocytic metabolism (Figure l)." Mea-
surements of net increases in red cell ATP support the contention that adenine loss
reflects nucleotide anabolism. Unmetabolized adenine inside red cells can quickly
January 1981 175
MEAN HCT=311
(n=12)
0 1 11 21 211 3s 42
DAYS OF 4C STORAGE
FIGURE 1. Plasma and red cell adenine concentrations during 6 weeks of whole blood storage
in CPDA-I at 4°C. The initial plasma adenine concentration is a theoretically calculated number
obtained by assuming instantaneous mixing of 17.3 mg adenine with the plasma from SO0 mf of
blood. The day 0 value was measured 30 rnin after phlebotomy and represents the plasma concen-
tration after initial plasma-red cell adenine equilibration has oc~urred.~*-"
The total value is the
calculated sum of adenine measurable in plasma and red cells.
egress from the cells if they are resuspended in a fluid (isotonic saline or plasma) not
containing adenine.z2.23
B. Metabolism of Adenine by Red Cells

Interest in purine metabolism, of which adenine is a part, has gained momentum
with the recognition that a group of congenital diseases including Lesch-Nyhan syn-
drome and gout are due to abnormal metabolism of purines. Since purine metabolism
has been extensively reviewed in recent year^,^^-^^ we will outline it only briefly here in
order to place red cell metabolism of adenine in proper biochemical perspective.
In most mammalian cell types, the mononucleotide inosine monophosphate (IMP)
is synthesized de novo from ribose-5-phosphate, glutamine, cysteine, and aspartic acid
(Figure 2). The other mononucleotides, adenosine monophosphate (AMP), xanthosine
monophosphate (XMP), and guanosine monophosphate (GMP) are formed by enzy-
matic conversions of IMP. These mononucleotides are readily converted to the nucleo-
tide di- and triphosphates which are utilized in nucleic a c i d synthesis and/or energy
transfer processes. IMP, XMP, and GMP are irreversibly dephosphorylated to the
corresponding nucleosides, while AMP is either dephosphorylated to adenosine or
(more readily) deaminated to yield IMP. The nucleosides of IMP, XMP, and GMP
can lose ribose reversibly to form the free purines hypoxanthine, xanthine, and guan-
ine, which are converted to uric acid in the liver. Murrayz6has shown that the amount
of uric acid excreted by the body is considerably less than the calculated production
7
Ribose S-P
AMP deaminasr
L
p.
DI
+
P
M-PR
Transferasa
IXonthine aridore
Axanthin. oridow
I 2.8-DoA 1
FlGURE 2. Overall scheme of mammalian purine metabolism. The reaction converting 5'-PRPP to IMP
(hatched line) consists of several steps representing the de ROVO synthesis of the purine ring from a ribose
phosphate base. This series of reactions does not occur in the human red cell. The human red cell does not
convert IMP to AMP, has only trace levels of xanthosine and xanthine, has no xanthine oxidase, and the
presence of AMP nucleotidase is questionable.
of purines due to mononucleotide degradation, indicating that the purines are largely
salvaged by the body for resynthesis into nucleotides. The salvage pathway has been
well d o ~ u m e n t e dand
~ ~ consists
-~~ of two enzymes: HGPR transferase,* which converts
hypoxanthine and guanine to their corresponding mononucleotides via reaction with
phosphoribosyl pyrophosphate (PRPP), and APR transferase,* which converts aden-
ine to AMP by reaction with PRPP (Figure 2). Since the conversion of nucleotides to
nucleosides is a unidirectional step in the catabolic pathway of nucleotide,la this path-
way is not reversible for purine salvage. As in most metabolic systems, the anabolic
or salvage pathway (e.g., HGP transferase or APR transferase) is in part different
from the catabolic pathway, providing the capability of independent control mecha-
nisms.
The human red blood cell contains no nucleic acids, cannot synthesize purines de
novo, nor convert IMP to AMP.'O The young red cell has a n adenine nucleotide pool
made up of ATP, ADP, and AMP in equilibrium with each other in the molar ratios
of 100:10:l.3*As the cells ages in vivo or in vitro (where the effect is more pro-
nounced), the nucleotide pool diminishes due to the irreversible deamination of AMP
to IMP which is further converted via inosine to hypoxanthine (Figure 2 ) . In 4°C-
stored whole blood, hypoxanthine accumulates linearly at a weekly rate of about 0.025
pmol/ml plasma or red cells.3z The only way the mature red cell can replenish its
adenine nucleotide pool is through the salvage of adenine using APR transferase to
yield AMP. The AMP reacts with ATP to yield 2'adenosine diphdsphate (ADP). Since
the original PRPP was formed from ribose-5-phosphate + ATP, the net stoichiometry
of the reaction is 1 adenine + 3 ATP yields 4 ADP. The rate limiting step in the
incorporation of adenine into nucleotides is the availability of the labile cosubstrate
PRPP. 13.33.34 PRPP concentrations are controlled by the enzyme PRPP synthetase
HGPR is hypoxanthine guanine phosphoribosyl, APR is adenine phosphoribosyl.

January 198 1 177
which combines ribose-5-phosphate and ATP to produce PRPP. This enzyme is allos-
terically inhibited by physiological concentrations of ADP, guanasine diphosphate
(GDP), and 2,3-diphosphoglycerate (2,3-DPG),3z.33but is partially relieved of this in-
hibition by elevation of inorganic phosphate leveb. Hershko, Razin, and Mager’” have
shown that ribose-5-phosphate is not rate limiting since red cells from severe glucose-
6-phosphate-dehydrogenase-deficient patients show no impairment in PRPP synthes-
izing a c t i ~ i t y . ’Because
~ of 2,3-DPG-hemoglobin (Hb) binding, these authors3’ have
speculated that in vivo the low oxygen tension (high 2.3-DPG-Hb binding) in venous
circulation would favor P R P P f o r m a t i ~ n . ’PRPP
~ synthetase in the red cell ordinarily
operates at a very small fraction of its maximal rate due to the complex combination
of inhibitors and activators discussed above.” The net effect of these inhibitors (and
activation by phosphate) is not yet well understood. The concentration of PRPP is
measurable throughout several weeks of in vitro blood Yip, Roome, and
B a l i ~ ~ ~ fractionated
.’’ red cells by age and observed increasing P R P P concentrations
with cell age and continued activity of P R P P synthetase even though the enzyme
seemed to undergo changes in molecular form. The presence of increased amounts of
P R P P during storage may offer an advantage for adenine supplementation. There is
also evidence in the literatureJ1 that 2.3-DPG can likewise inhibit the activity of APR
transferase in human erythrocytes. Thus, 2,3-DPG seems to play an important role in
regulating the rate of adenine conversion to AMP.
The converse effect of adenine o n the concentration of red cell 2,3-DPG during
blood storage has also been examined. Many studies have shown that red cell 2,3-DPG
concentrations fall rapidly in blood stored in CPD (Table 1). usually approaching zero
by 21 to 28 days of tora age.^ Early studies with ACD and ACD-adenine demonstrated
that the presence of adenine was associated with a faster drop of 2,3-DPG than in
ACD a l ~ n e . More ~ ~ . recent
~ ~ studies of red cells preserved in CPD and CPD-adenine
also indicated that in CPD-adenine 2,3-DPG fell more rapidly than in C P D units.
CPD-adenine was superior to ACD-adenine in maintaining 2,3-DPG.z3.‘0 Moore et
al.“ have studied the effect of various adenine and glucose concentrations on red cell
2,3-DPG in whole blood and packed red cells at controlled hematocrits. They con-
firmed that adenine (0.25 or 0.50 m M i n blood) caused a slightly faster drop in 2,3-
DPG, but when the data were analyzed by repeated measures analysis of variance and
a conservative multiple comparisons test, the difference between C P D and CPDA- 1
was significant (p < 0.05) only on day 14 of storage (Figure 3). A t all other storage
times, measured weekly from 0 to 6 weeks, the slightly lower 2.3-DPG levels in aden-
ine-containing units were not significantly different.
The question of whether or not adenine affects the deamination of AMP to IMP is
unanswered. If adenine were to inhibit this deamination reaction, it would amount to
a secondary mechanism whereby adenine maintained red cell ATP. Ambrus et aL4’
have attempted to assay AMP deaminase in the presence and absence of adenine. They
used fresh red cells, lysed them by freeze-thaw, and incubated the hemolysates at 37°C
until ATP and ADP disappeared. Under these circumstances, the authors claimed that
any further loss of AMP would be due to AMP deaminase and the change in AMP
over a fixed time period (3 hr, 37°C) should be a measure of this enzyme activity.
Using this method, Ambrus et aL4*showed a 56% inhibition of A M P deaminase by 1
mMadenine. Their assumption that AMP loss is a measure-of deaminase activity is
subject to question since AMP itself is a compound which may undergo spontaneous
decomposition. Moreover, they assumed that there was no AMP nucleotidase activity
which would convert AMP to adenosine. While the existence of the nucleotidase has
not been reported in recent reviews on red cell purine m e t a b o l i ~ m , ~Tsuboi ~ . ~ ~ and
.~~
Hudson4‘ in 1953 described a human red cell “ p h o ~ p h o m ~ n ~ e ~ t e rwhich a ~ e ” could
convert yeast adenosine phosphate t o free adenosine. BartletP addressed the issue of
PACKED CELLS HCT=70
100
90
80
5 70
Y
Y)
+I
s
-o 60 - A1
..............A 2
’
g
50
--CPD
4
2 40
z
a
2 30
20
10
0 7 14 21 28 35 42
DAYS
FlGURE 3. Red cell 2.3-diphosphoglycerate (2.3-DPG) during 42-day storage (4’C)

of packed red cells in three anticoagulants: CPD, CPDA-I, and CPDA-2 (n = 5 each).
2.3-DPG is expressed as percentage of the prestorage value.
how adenine contributes to ATP maintenance by using “C-labeled adenine in two units
of ACD blood stored for 6 weeks. From measurements of the concentrations of all
red cell nucleotides and purines at 2-week intervals, he concluded that the bulk of the
adenine enters the nucleotide pool, “ruling out the possibility that inhibition of nucleo-
tide (AMP) breakdown plays a significant part in the adenine effect of stored blood.”
The study of Bartlett3zdoes not seem to rule out modest levels of deaminase inhibition,
particularly during the first 2 to 4 weeks of storage. Lian and H a r k n e s ~ ‘have
~ studied
the kinetic properties of human red cell AMP deaminase after extraction and 500-fold
purification. They found that the enzyme was activated by ATP and monovalent cat-
ions, but that adenine had no effect on the activity. The kinetic studies of Lian and
H a r k n e s ~ ‘d~o not provide a definitive answer to the question of adenine effects on
the deamination of AMP to IMP since their enzyme purification procedure may have
altered the properties of AMP deaminase. This possibility was also suggested by Sasaki
et a1.,46 who studied the kinetics of AMP deaminase under simulated intracellular con-
ditions and found different activatiodinhibition effects with ATP and 2,3-DPG than
those seen by Lian and Harkness.‘*
January 1981 179
Two recent ~ t u d i e s ~ ' concerning

.'~ the incorporation of adenine into intact human
red blood cells provide kinetic data on the conversion to nucleotides during controlled
incubations. Dean and Pel-rett)' studied the uptake of ' ' C - a d e n ~ ~ i n by e washed red
cells suspended in buffered salt solutions at 37°C. They confirmed the work of oth-
ers17-20 2 2 . 2 3 . 2 6 - 2 9 in showing the rapid uptake of adenine into nucleotides, the deamina-
tion of adenosine to inosine and its conversion to hypoxanthine (instead of conversion

to ATP), and the activating effects of elevated phosphate levels on the purine salvage
pathways. They3' also reported data supporting the contention that the rate and extent
of these reactions was greatly influenced by the conditions of the experiment such as
the composition of the suspending medium, the nature of the gas equilibration, etc.
Purine uptake into nucleotides has also been measured by de Bruyn and Oei" using
washed red cells suspended in buffer at 37°C. They also confirmed rapid uptake of
hypoxanthine, guanine, and adenine and conversion to their corresponding nucleo-
tides. de Bruyn and Oei" showed a two-phase uptake dependent on adenine concentra-
tion: at low concentrations, uptake paralleled conversion to nucleotides, while at
higher concentrations, uptake continued to increase in a linear manner (but with lower
slope) while the nucleotide-forming pathways appeared saturated. In addition, they"
demonstrated that exogenous addition of P R P P to the system resulted in extracellular
nucleotide formation with all three purines, which suggested that HGPR transferase
and APR transferase are membrane enzymes which may couple the properties of pur-
ine transport and nucleotide formation.
Our analysis of the available literature on adenine uptake and metabolism in human
red cells has led us to draw several conclusions. The transport of adenine into the red
cell is rapid. Some investigator^'^.^^^^^.^^ believe adenine transport to be by facilitated
diffusion, although this has not been clearly ~ u b s t a n t i a t e d Differing
. ~ ~ ~ ~ rates and rel-
ative amounts of uptake have been reported in the literature,16.20~22~24.25~31~47 but this is
probably due to the large variation in experimental conditions used by different inves-
tigators. These variations include choice of red cell suspending medium (plasma,
buffer, saline, etc.), temperature, concentrations of purine, glucose, and phosphate,
time, age of cells, pretreatment of cells, and method of analysis. The controlled studies
of Dean and Perrett" confirm the large effects which these variables have on purine
uptake. The studies of red cells suspended in plasma at 4°C22-25J2 most clearly dem-
onstrate the utilization of adenine as part of a n anticoagulant preservative solution for
blood storage. The main metabolic fate of adenine in the red cell is to combine with
P R P P under the control of APR-transferase to form AMP. The AMP is in equilibrium
with A D P and ATP, effecting a net increase in the concentration of the adenine nu-
cleotide pool. Under blood banking conditions, this reaction occurs slowly over several
weeks, resulting in a significant contribution t o the maintenance of red cell A T P con-
centration. A proposed secondary but minor mechanism of adenine action in the red
cell is to to inhibit A M P deaminase from converting A M P to IMP;31.42.44-46 however,
the data on this issue are conflicting and sketchy.
C. Transport of Adenine into Platelets

Adenine transport into platelets exhibits kinetic properties which are mechanistically
similar to adenine transport into red cells. For example, uptake of adenine at low
concentrations appears to be carrier mediated as evidenced by a saturable transport
process with Michaelis-Menten properties and inhibitory effects of other purines and
n u c l e o ~ i d e s ~(Figure
* - ~ ~ 4). Transport of adenine into platelets occurs with a Km o f
159 n M a n d a Vmmr of 100 k 10 pM/min/109 platelets in citrated plasma at 37°C.50
D. Metabolism of Adenine by Platelets

The metabolism o f adenine and its nucleotides by platelets has been reviewed else-
Extracellular Pool N o n g r a n u l a r Pool Granular Pool
PRPP3
+ 4
Adenine -c AMP
lnosine \ *
Hypoxanthine7 + PRPP
Xonthine
I
Uric Acid
FIGURE 4. Schematic of adenine transport, compartmentalization, and metabolism in human platelets.

( I ) Michaeiis-Menten transport; Vm = 100 pmol/min/IO’ platelets and Km = 159 nmol at 37°C. (2) path-
way has not been shown; adenine is rapidly converted to nucleotides intracellularly. (3) rate of formation
of PRPP is unknown for platelets, but is probably slow. (4) AMP can also be produced from adenosine,
( 5 ) Transport is poorly characterized; studies in rabbits suggest zero-order kinetics with an estimated Vm y
5.3 pmol/hr/lO’ platelets at 37°C.” (6) ATP is partially hydrolyzed to ADP resulting in an intragranular
ATP:ADP ratio of 2:3 in human platelets,” (7) the role of this pathway is thought to be of major impor-
tance. and (8) degradation of hypoxanthine is accomplished extracellulariy: = pathway has not been
demonstrated; ** = pathway kinetics have not been studied.
here.^'.^^ Platelets, like erythrocytes, are unable to synthesize purine nucleotides de

concepts of adenine metabolism include three pathways whereby plate-
I I O V O . ~Current
~
lets can obtain purine nucleotides. Platelets contain adenine phosphoribosyltransfer-
ase49and therefore can synthesize adenine nucleotides directly from adenine. They can
also use exogenous adenosine via adenine k i n a ~ e . ‘In
~ addition, platelets can convert
hypoxanthine to AMP via IMP with hypoxanthine phosphoribosyltransferase (Figure
2 ) v s 3Although the relative contribution of each pathway is unknown, the availability
of hypoxanthine to the platelets would make the hypoxanthine salvage pathway attrac-
tive for consideration as a major Evidence has been p r e ~ e n t e d ~ ’ .that
~‘
platelets can reutilize intracellular hypoxanthine.
E. Subcellular Distribution and Metabolic Function of Adenine Nucleotides

In addition to maintaining the metabolic integrity of platelets..adenine nucleotides
contribute a significant amount of energy for functions involved with hemostasis. For
example, platelet a d h e ~ i o n , ~the
~ . ~“release
’ reaction”,5a-60and “ a g g ~ e g a t i o n ” ~ ’ -all
~’
require ATP. Clot retraction also appears to be dependent upon energy derived from
adenine nucleotide~.~’ While energy dependence is well accepted for release, aggrega-
tion, and clot retraction, there is disagreement regarding the biochemical basis of adhe-
sion.55-57.60.64-66
Conflicting reports may be explained by the difficulty of separating adhesion from

January 1981 181
other clotting events (i.e., aggregation) and by variation in test systems used by inves-
t i g a t o r ~ The
. ~ ~early
~ ~ phase of adhesion, at least, does not appear to be energy de-
pendent since formalinized platelets will adhere to polylysine-coated glass surfaces and
can react to agglutinating agents such as ristocetin-vonWillebrand factor, bovine V I I I ,
or p ~ l y s i n eeven
, ~ ~ though formalin decreases the amount of ATP.70
The distribution of adenine nucleotides within platelets shows distinct subcellular
compartmentalization. Holmsen, Day, and Storms8 have proposed the concept that
adenine nucleotides reside in three distinct compartments of “ p o ~ l s ” .Using
~ ~ radio-
labeled nucleotides, these investigatorss9 found a labeled fraction and an unlabeled
fraction capable of being released when the platelet was stimulated with thrombin.
The unlabeled pool had a low ATP:ADP ratio and constituted about 60% of the total
platelet adenine nucleotide!;. This pool was part of the storage granules5’ and since it
did not take up any additional label within 2 hr of the start of incubation, it was
considered an inactive pool insofar as metabolism is concerned. The metabolically ac-
tive pool was found in cytoplasm. mitochondria, and membranes.68 During release
induced by collagen or thrombin, the metabolically active pool lost about 25% of its
adenine nucleotides by conversion to IMP. Holmsen and colleagues5’ 5 8 considered the
ATP thus lost to be a third pool which is in equilibrium with the metabolically active
pool. In light of current concepts of nucleotide t~rnover,~’.’’ it appears the third pool
may represent a limited shift in the metabolically active pool from high energy charged
nucleotides to lower forms. Hence, the ATP consumed results from a nucleotide im-
balance. The imbalance is the aftermath of utilization in excess of production of ATP
for activities such as the secretory event.70
Adenine nucleotides in the metabolic pool may play a regulatory role in platelet
metabolism. This action wlould allow regulation to occur in systems where substrate
limitations make product feedback control inappropriate. 72 Additionally, such control
provides precise response characteristics such as are found with regulatory enzymes,
even when ATP concentration varies within a small range.73 ATP conservation is a
major feature of metabolic regulation in all ATP utilization reactions. 74
The nature of the granular nucleotides has been extensively inve~tigated.~’-~’ The
flow of adenine nucleotides into this releasable pool has been shown to be dynamic in
nature, but with different kinetics than the metabolically active pool. Figure 4 is a
schematic representation of this relationship. Reimers, Mustard, and P a ~ k h a mhave ~~
shown that in rabbit platelets the metabolic pool slowly establishes equilibrium with
the nonmetabolic (granular) pool. Most probably the nucleotide being transported into
the granular pool is ATP.54’’ Human platelets show a similar uptake by the granules.54
ATP once delivered to the granule may not remain metabolically inert as exchange
with the nonmetabolic pool seems likely. Reimers, Packham. and Mustards4 reported
evidence that A T P is partially hydrolyzed to A D P since prolonged incubation of plate-
lets containing “C adenine nucleotide results in a change in intragranular ATP:ADP
ratio.
F. Biochemical Effects of Adenine on Platelets under Storage Conditions

Adenine nucleotide metabolism may be influenced by several factors during platelet
storage which are not opera.tive in vivo, but which play an important part in cell integ-
rity and metabolism. Initial hydrogen ion concentration, temperature of storage, total
platelet count, volume of preservative medium, duration of storage, agitation during
storage, and hydrogen ion accumulation are some of the factors known to influence
the storability of platelets.;‘8 The platelet adenine nucleotide levels have been studied
in relation to temperature of tora age.'^-'' Although techniques of assay differed, there
was general agreement by Kim and Baldini79 and Filip, Eckstein, and SibleyE0that
adenine nucleotide levels remained above 70% of prestorage values throughout 72 hr
storage at 4°C. During 22°C storage, ATP and ADP levels were erraticBaand appeared
related to pH. When the pH was maintained above 6.85 during 22°C storage, nucleo-
tide levels were no different from those of units stored at 4°C. This may reflect pH
dependency of certain enzymes in the nucleotide pathway such as adenine phosphori-
bosyltransferase and hypoxanthine phosphorib~syltransferase.~~ The loss of activity of
the latter enzyme would explain observations by Filip, Eckstein, and Sibleyso that all
radiolabelled ATP was converted to hypoxanthine. Storage for up to 4 hr at either
4°C or 22°C does not deter adenine uptake or ATP production.@’
On theoretical grounds, adenine added to the platelet preservative could be benefi-
cial assuming nucleotide enzyme activity and other substrates (such as PRPP) could
be maintained. The capability of the platelet t o utilize exogenous adenine over a period
of time is questionable, however. Holmsen and R o ~ e n b e r g *have
~ shown that adenine
phosphoribosyltransferase in platelet lysates was unstable at 37”C, but stable at 4°C
for at least 24 hr. In addition, the concentration of P R P P present in platelets is limited
(estimated at 1 p M in crude lysates). P R P P is produced from 5-ribose-phosphate and
ATP by P R P P synthetase. The latter enzyme cannot be detected in crude l y ~ a t e s . ~ ~
Despite these potential biochemical limitations. there is some evidence that platelets
can benefit by the presence of exogenous adenine. Odake and Ambruses2added high
concentrations of adenine ( I mM) to platelet-rich plasma in both C P D and ACD pre-
servatives. At the end of 3 days, the aliquots stored with adenine had 15% more nu-
cleotides than prior t o storage and 18% more than aliquots stored 3 days without
adenine. A calculated AEC was also maintained.* Ader1ine-8-’~Cadded to platelet-rich
plasma harvested in ACD was incorporated into adenine nucleotides after 3 days of
storage. Unfortunately, the study was small (n = 4), storage was not in accordance
with current standards, and pHs were not reported. These data support the contention
that platelets appear, a t least under certain conditions, to be able to utilize exogenous
adenine during storage.
111. TOXICOLOGY
A. Metabolism and Pharmacokinetics of Free Adenine in Man

Data relevant to the fate of adenine administered to man have been provided in
seven detailed investigations. Roth et al.04 infused adenine, 5 or 10 mg/kg, into 12
normal volunteers and measured adenine and adenine oxymetabolite concentrations in
plasma and urine. Bartlett” reported plasma, erythrocyte, muscle, and urine I4C-con-
centrations and chromatographic analyses following injection of 8-I4C-adenine 10 mg/
kg, into one normal volunteer in two separate experiments. DeVerdier et aLBSstudied
plasma and urinary adenine and adenine-metabolites in two healthy subjects given ad-
enine, 3.4 to 4.5 mg/kg intravenously and 3.6 mg/kg orally. Urinary excretion of la-
beled uric acid following oral ingestion of 8-Y-adenine by two normal and three gouty
subjects was examined by Seegmiller et a1.86One healthy subject who received an intra-
venous injection of adenine-8-I4C was studied by Wyngaarden et al.*’ Bishop8@meas-
* ATP conservation and adenine nucleotide regulation has been expressed as the adenylate energy charge
(AEC).*I
AEC = (ATP + %ADP)/(ATP + ADP +- AMP)

The calculated AEC was derived from values for adenylates done by chemical rather than radioisotope
assays and, as such, does not solely reflect metabolic pool activity. However, time changes can probably
be roughly estimated. The use of AEC to evaluate adenylate control of metabolic regulation during
storage of platelet concentrates may not be appropriate since maintenance of the AEC required the
presence of adequate adenylate kinase to drive the system.” The function of adenylate kinase may be
impaired duririg storage.
January 1981 183
wed labeled blood uric acid, nucleotides, and ribonucleic acids following injection of
tracer doses of 8-'*C-adenine into two healthy elderly males. Kreugera9reported plasma
and urinary adenine and 2,3-dihydroxyadenine concentrations in four newborn babies
measured during and after exchange transfusions with CPD-adenine blood (adenine
dose -4 mg/kg). Each of t.hese reports lacks complete pharmacokinetic analysis, but
taken together they yield a, consistent view of adenine disposition, metabolism, and
pharmacokinetics (vide infra). de Verdier, Groth, and Westrnan9O have proposed a
mathematical model for simulation of human adenine metabolism and pharmacoki-
netics during blood transfusion. Their model is physiologically plausible and incorpo-
rates appropriate adjustments for hemorrhagic losses. However, it is overly compli-
cated for estimating model parameter values from data available and therefore
assumed values must be relied upon for most parameters. The few parameter values
estimated from the data are imprecise and highly dependent upon model assumptions.
Hence, confidence in this model awaits further data to test its adequacy.
Measurements of basal plasma adenine concentration in normal man range from
4.7 to greater than 150 ng/'m1.8s.91.9z The wide range of reported values probably re-
flects differences in assay techniques applied to the measurement of this purine base
which is normally present in trace concentrations in plasma. Transient, locally high
concentrations of plasma adenine may develop during intravenous infusion of adenine
(highest reported value - U .3 pg/mPa5). However, the rapid disappearance of adenine
from plasma precludes intravascular precipitation due to exceeding its aqueous solu-
bility, which is 1700 mg/1 at 36.6°C.93The extent of plasma protein binding of adenine
has not been reported.
Plasma disappearance of adenine following intravenous infusion is best described
by a sum of exponentials. Estimates of a single disappearance half-life of plasma ad-
enine range from a few seconds to a n These differences are probably due
to variations in adenine infusion rates, sampling times, and data analytic procedures.
When plasma adenine concentrations are measured beginning 5 min after a rapid intra-
venous infusion, the predominant disappearance half-life is 15 to 25 min. By 5 min
postinfusion, however, a large portion of the adenine has exited the vascular system.
Bartlett13 suggested that adenine loss from plasma occurs in two phases: a very rapid
phase (within the first few seconds), which accounts for 80 to 90% of injected dose,
followed by a phase with a half-life of approximately 1 hr. The rapid phase indicates
extravascular distribution, while the slower phase reflects uptake and metabolism of
adenine by tissues.
The urinary excretion rate of adenine under basal conditions is 0.,3to 6.2 mg/day
in healthy volunteers and in gouty and leukemic Up to 25% of intra-
venously administered adenine appears as adenine and adenine derivatives in urine
within 6 to 8 hr after infusion.13.84.85
Free adenine is the predominant excretion product
during the first 3 hr, while the oxymetabolites, 8-hydroxyadenine (8-OA) and 2,8-dih-
ydroxyadenine (DOA), begin to accumulate after a short time lag. Excretion of these
compounds is largely complete by 24 hr after administration. Thereafter, or ''C
from adenine labeled in the adenine-8-C position has been detected in urinary uric acid
for periods up to 3 months following oral or intravenous administration. Excretion as
uric acid was reported to be initially about 1.5% of the administered dose per day and
decreased to 0.5% 3 months later. 'J.86 These observations are-consistent with the con-
cept of immediate glomerular filtration of free adenine, delayed excretion of oxyme-
tabolites due to time required for biotransformation of adenine, and ultimate excretion
of the remaining 75% which entered the nucleotide pool in the form of uric acid.
Figure 5 summarizes what is known of the fate of infused adenine in adult
humans. 13.84-88Small but similar fractions of intravenously administered adenine are
excreted as unchanged adenine and adenine-oxymetabolites, 8-OA and DOA. The ox-
DISPOSITION OF INFUSED ADENINE IN M A N
FIGURE S. Schematic of the disposition of free infused adenine in man. XO is xanthine oxidase.
ymetabolites have been shown to result from the action of xanthine oxidase on adenine
and 8-OA.99-i01 This action probably takes place to a large extent in the liver, the high-
blood-flow organ with the highest amount of xanthine o ~ i d a s e . ’ 8-OA ~ ~ . ~is~ more
~
soluble in plasma water (aqueous solubility = 75.6 mg/f at room t e m p e r a t ~ r e ) ”than
~
DOA (aqueous solubility = 1.53 2 0.04 mg/f at 37°C). Ios However, DOA is prevented
from intravascular precipitation because of its binding to proteins such as serum al-
bumin. SternIo6has shown that DOA affinity for human albumin exceeds that of ad-
enine’O’ and uric acid.Ios However, data are not available which would allow a quan-
titative assessment of the risk of intravascular DOA precipitation on the basis of
binding capacity of circulating albumin mass and DOA production from a given load
of adenine. Solubility of DOA in urine also exceeds its water solubility considerably:
in vitro urine solubility at 37°C is 40.4 -+ 3.3 rng/f, while in vivo urine solubility is
greater than 96 mg/f .Ios The bulk of infused adenine is taken up by a variety of tissues
and incorporated into adenine nucleotides. Bartlett” documented incorporation of
into AMP, ADP, and ATP of red blood cells within 2 hr following infusion of 8-I4C-
adenine and detected it in ATP of gluteus maximus muscle 4 days later. The highly
efficient salvage process to which purines of the nucleotide pool are subjected results
in a slow loss of the position-8 carbon which entered the nucleotide pool. This occurs
because of a combination of salvage pathways and the AMP deaminase reaction con-
verting AMP to IMP and then hypoxanthine, yielding urinary uric acid (Figure 2).
Small quantities of “C (originating from S-I‘C-adenine) can be detected in urinary
hypoxanthine, xanthine, guanine (trace), and 7-methylguanine (trace) for up to be-
tween 7 and 10 days after injection.” in uric acid continues to be excreted for
several months.13 The potential addition to uric acid excretion, however, appears t o
be too gradual to increase it t o a level which would result in plasma o r urinary precip-
itation of uric acid crystals.
The metabolic pathway of infused adenine depicted in Figure 5 applies to healthy
or gouty adults. The fate of adenine in nonadult subjects has been reported only in
January 1981 185
patients suffering from hemolytic disease of the newborn. Kreugern9demonstrated in

these neonates who underwent exchange transfusion with CPD-A blood that plasma
adenine disappearance is rapid compared to the rate of disappearance in adults. More-
over, the fraction of infused adenine appearing in urine as unchanged adenine (less
than 0.4%) or DOA (less than 1.1070) is considerably less in the neonate than in adults.
Kreugers9 attributed these differences to increased activity of APR transferase and
xanthine oxidase in the newborn.
B. Recipient Toxicity from Adenine in Blood Products

Blood stored with a CPD-adenine preservative contains both free adenine in plasma
and diffusible adenine in red cells. For toxicological considerations the sum of these
two sources of adenine is considered the “dose” of free adenine to which a recipient
is exposed during transfusion. Moore et a1.” 23 have shown that the distribution of
adenine between plasma arid red cells as well as the disappearance kinetics of intra-
and extracellular adenine throughout 21- to 42-day blood storage in CPDA-1 is similar
to blood stored in ACD-A.” Figure 1 indicates than an exponentially smaller propor-
tion of the original adenine in the preservative is available for systemic distribution
upon infusion; less than 30% of the original 17.3 mg adenine is available after 21-day
whole blood storage.” A similar study with whole bloodz3showed that less than 13%
of the original adenine preservative content is available after 35-day and less than 11 070
after 42-day storage. Blood stored as concentrated red cells contains less than 50% of
the free adenine in whole blood due to the removal of adenine-rich plasma during
preparation of concentrated red cell units.
Nephrotoxicity is the principal acute adverse effect in animals and man resulting
from adenine administration jog- and is thought to be related to precipitation of the
poorly soluble metabolite of adenine, DOA, in the urinary t r a ~ t . ~Warner”~ ~ . ’ ~re-
~
viewed published and unpublished studies of adenine toxicity in animals. We cite here
results of several investigations which typify the more extensive compilation by War-
ner. The LDso for rats and mice was in the range of 200 to 350 mg/kglo9when adenine
was administered i.p. I.V. administration of adenine, 50 mg/kg. caused neither renal
dysfunction nor morpholclgic lesions in normal and dehydrated rats and mongrel
dogs; l o p 25 mg/kg was equally well tolerated in unilaterally nephrectomized rats and
normal Dalmatian dogs.”O EIevation of blood urea nitrogen (up to 40 rngVo) for 24
hr after administration of adenine was the only abnormality noted at doses between
50 and 100 mg/kg in rats.109Baboons given 25 mg/kg i.v. during hemorrhagic shock
showed no differences in renal function after recovering from shock when compared
to nonadenine-treated animals. Chronic i.v. administration of adenine, 10 mg/kg/
day for 15 days, was well tolerated in Dalmatian dogsto9as well as in beagle dogs given
10 rng/kg per five to six times per week i.v. for 3 Monkeys (Maccara irus)
showed no evidence of toxicity when given adenine 10 mg/kg/day i-v. for 5 days.11s
N o teratologic sequelae were found in pregnant HoItzman rats and New Zealand rab-
bits administered adenine 50 mg/kg during the 6th to 15th day of gestation.”’
Auditore et a1.116have reported that 50 mg/kg bolus i.v. injections of adenine sulfate
induce a mild granulocytosis in rabbits. Shields et al. ”’ performed leukocyte counts
and peripheral blood morphologic examinations in a human study involving rapid ad-
enine infusions. Quantitative results were not reported, but -the authors commented
that there were no hematological abnormalities. The adenine dosage used in the latter
study was an order of magnitude lower than that in the study of Auditore et a1.Ir6
Dose and species differences and use of the adenine sulfate salt may account for the
lack of granulocytosis in the human study.”’ This aspect of possible toxicity may need
to be investigated further iif unexplained granulocytosis following transfusion is en-
countered.
Only one human case of proven adenine toxicity has been rep0rted.l" In this patient,
approximately 71.4 mg/kg (assumes body weight of 70 kg) per day for 5 days was
administered orally in an attempt to treat pernicious anemia. The patient developed
severe uremia, but recovered almost completely (residual blood urea nitrogen of 38
mg was the only abnormality).
One case of possible toxicity resulting from administration of an adenine-containing
blood product has been reported. 1 1 8 A postoperative cardiac surgery patient was ad-
ministered 118 units of ACD-A (adenine 95 mg/kg) during a 5-day period. Evidence
for adenine toxicity included impaired renal function and postmortem demonstration
of renal calculi which were morphologically similar to DOA. However, this patient
had transient serum creatinine elevations prior to receipt of ACD-A blood and experi-
enced multiple episodes of hypotension and shock in the postoperative period. In spite
of the combined effects of shock and possible adenine toxicity a diuresis was main-
tained until just before death. Death was attributed to hemorrhagic shock."8
Numerous studies attest to the safety of i.v. administration of adenine to humans
in doses up to 15 mg/kg (equivalent to 60 units of fresh CPDA-1 whole
b l ~ ~ d ) No. immediaten4 ~ ~ ~ or ~long-term
~ ~ ( 5~ year ~ follow-up)121
~ ~ ~ toxicity
~ ~ has ~ - ~ ~ ~
been detected despite extensive renal function tests. Roth et al.84for example, assessed
glomerular function (creatinine clearance, urinary protein excretion), proximal tubular
function (urinary excretion of glucose and 15 amino acids), and distal tubular function
(maximal urinary acidifying and concentrating ability) and found n o adverse effect of
i.v. infusion of adenine, 5 and 10 mg/kg in normal volunteers. Complete exchange
transfusions with adenine-fortified blood in neonates had no detectable physical, bio-
chemical, or renal effects acutely and up to 5 years later."'
Consideration of an upper adenine dose limit has relied upon the demonstrated
safety of rapid i.v. infusion of 10 to 15 mg/kg.9.105.118 On this basis, Simon9 suggested
that up to 30 fresh, 0.5-mMadenine (e.g.. ACD-A or CPD-2A) whole blood units or
60 fresh, 0.25-rnM adenine (e.g., CPDA-1) whole blood units could be safely trans-
fused. However, it must be recognized that the safety of this dosage was established
in healthy adult volunteers. Recipients of adenine blood products who are hypotensive,
dehydrated, o r have compromised renal function may be at increased risk for precipi-
tation of DOA crystals and subsequent renal parenchymal damage. However, mere
demonstration of DOA crystals in the urine of a recipient of adenine blood is not
sufficient evidence for adenine toxicity. Like uric acid crystalluria, DOA crystals may
precipitate from a drop of urine placed on a cold microscope slide, although the com-
pound was soluble in urine at body temperature. PeckIos suggested that up to 25 fresh
CPDA-1 whole blood units can be given over a relatively short period of time without
formation of DOA crystals in the urine in vivo.
C. Effects of Adenine on Components of Stored Blood

Although it appears that free adenine has minimal toxic effects on organs after in-
fusion, various concentrations of adenine and/or prolonged exposure to adenine may
be toxic to cells and plasma components during blood storage. A negative effect of
adenine on erythrocyte 2,3-DPG maintenance has been discussed in the section on red
cell metabolism and elsewhere. IZ7 Other aspects of erythrocyte metabolism appear un-
affected by the presence of adenine.
Adenine toxicity to platelets has until recently not been explored. Katz et al."' re-
ported on the effect of CPDA-1 preservative on platelet concentrates prepared under
routine blood bank conditions. They found no differences in pH or morphology after
72 hr of storage in comparison with C P D controls. However, the ability to recover
from osmotic stress, proposed as a test to predict platelet recovery in V ~ V Owas
, ' ~im-
~
paired in those units stored with adenine. Katz et al.lZs further demonstrated that the
January 1981 187
inhibition of osmotic recovery could be induced directly in a dose-dependent fashion

by incubating platelets for 30 min with adenine. Bolin et al.I3O studied effects of two
concentrations of adenine (0.25 and 0.5 mM) and three concentrations of glucose (125,
175, and 200% of the glucose content in standard CPD) on platelet concentrates stored
for 72 hr at 22°C. When compared with C P D controls, no differences in pH could be
detected, but morphology was better maintained in preservatives containing adenine
than in controls. Unfortunately, studies of both Katz et a1.128and Bolin et al.I3O lacked
functional platelet evaluations or correlation with in vivo platelet performance. Plate-
lets have been prepared from whole blood units collected in ACD-A in S ~ e d e nThe .~
presence of adenine presented no additional difficulty in platelet concentrate prepara-
tion. Scott and S l i ~ h t e r ' ~
have
' evaluated the in vivo viability and function of platelets
stored for as long as 72 hr in CPDA-I. In 16 normal volunteers, recovery of infused
platelets was 50 2 14%, while mean life span of platelets was 7.3 2 1.5 days. Function
was determined by template bleeding times in 12 thrombocytopenic patients. Improved
bleeding times were obtained in seven patients who showed a rise in platelet count after
transfusion. Five patients with minimal or n o rise in platelet count after transfusion
showed no improvement in bleeding time. The authors concluded that platelets pre-
pared and stpred in CPDA-I were comparable to platelets prepared and stored in CPD.
The effect of adenine on stored leukocytes has not been studied. Both lymphocytes
and granulocytes are capable of metabolizing exogenous adenine. Leukocytes have
APR transferase activity. 132 This suggests that leukocytes could utilize the free adenine
used in blood preservative systems. Since leukocyte preservation is not a concern in
routine blood banking prac:tices, the only consideration would be theoretical; for ex-
ample, white cell toxicity with liberation of toxic substances or enhanced microaggre-
gate formation.
The effect of adenine on plasma proteins is an important consideration for blood
banks because of the fractionation of whole blood for the production of plasma deriv-
atives. Of prime importance is the status of Factor VIII in fresh frozen plasma and
cryoprecipitates. Graybeal, Mooreside, and L ~ n g d e l l studied
'~~ cryoprecipitates pre-
pared from CPD- and ACD-preserved whole blood which had been supplemented with
approximately 0.5 m M adenine. They found no effect of adenine on recoverable yield
of Factor VIII. However, the study lacked critical statistical analysis and did not eval-
uate the effects of other concentrations of adenine and added glucose. The same au-
t h o r ~ had
' ~ ~shown earlier that fresh frozen plasma stored at -30°C for 3 months
exhibited no difference in Factors V , VIII, IX, or X when prepared fresh from adenine-
supplemented (0.5 mM) C P D or ACD blood. N o differences which could be ascribed
to C P D , CPDA-2, and CPDA-3 were detected (n = 10, each preservative) by analysis
of variance ( p > 0.05) in Factor VIII activity in fresh plasma or in plasma stored for 8
hr at room t e m p e r a t ~ r e . Ambrus
~'~ et studied plasma proteins and coagulation
factors in blood drawn into six ACD and ACD-A units. They could discern no differ-
ence between the two preservatives in freshIy drawn plasma o r cryoprecipitated plasma
in the concentrations of albumin, globulin, or coagulation Factors I, 11, V, VII-X, and
XIII. These also studied the coagulation and fibrinolytic system in CPD,
ACD, and adenine-supplemented C P D and ACD blood stored for 42 days. The time-
dependent loss of coagulation factors was similar in all four anticoagulants. Plasmin-
ogen and antiplasmin levels were also measured, and again fio differences were de-
tected which could be ascribed to the preservative media. Because these components
of the fibrinolytic pathway were measured by bioassay and sample sizes were small,
the results are questionable. Euglobulin clot lysis times were also interpeted as showing
no change among anticoagulants. However, there does not appear to be any evidence
that stored blood, fresh frozen plasma, or frozen blood has any clinical fibrinolytic
activity. 135.136
D. Effects of Adenine on Plasticizer Leaching and Metabolism

The plasticizer di-Zethylhexyl phthalate (DEHP) as well as its metabolite mono-2-
ethylhexyl phthalate (MEHP) accumulate in plasma of blood stored in polyvinylchlor-
ide bags.'" Although no adverse effects attributed to the unavoidable infusion of
DEHP and MEHP have been identified after transfusion into patients, concern over
this possibility has inspired a considerable body of literature.
Influence of adenine on plasticizer accumulation may arise from two sources: in-
creased contact time between blood and plastic due to extension of blood storage du-
ration and adenine effects on DEHP metabolism in plasma. Plasma DEHP and MEHP
concentrations have been reported to increase linearly with time during 21-day storage
in C P D or ACD preservation system^.'^^.^^^ Peck et a1-14'documented that DEHP and
MEHP continue to accumulate in blood stored beyond 21 days in CPD or CPDA-I,
but showed that the accumulation rate declines with increasing storage time. Variation
in MEHP accumulation rates measured in blood stored in bags made by two manufac-
turers were attributed to differences in topographical properties of the plastic surfaces
in contact with the blood.'41
Cole et al. have presented data suggesting that adenine in CPD-adenine preserva-
tives reduces both the leaching rate of DEHP and the rate of hydrolysis of DEHP to
MEHP during blood storage. They demonstrated in a partially purified plasma enzyme
preparation that adenine exerts an inhibitory effect upon the plasma enzyme(s) respon-
sible for DEHP hydrolysis. This could be advantageous since MEHP is considered to
be potentially more toxic than DEHP, '41 but the effect is quantitatively small and may
have little clinical consequence. Peck et al.I4' did not find differences in DEHP leach-
ing rate attributable to preservative content of adenine, but their study was confounded
by the fact that experiments were performed in bags made from different plastic for-
mulations.
IV. CLINICAL USE OF ADENINE-PRESERVED BLOOD
A. Use of Adenine-Preserved Blood in Europe

Large-scale clinical evaluation of blood stored with a preservative solution contain-
ing adenine commenced more than a decade ago in Sweden and Germany. Akerblom
et al.,"' at the University Hospital in Uppsala, Sweden, reported that the number and
type of reactions observed in 5595 transfusions of ACD-A (storage time 0 to 35 days)
were no different than those observed in 4519 transfusions of standard ACD blood
(storage time 0 to 21 days) given during two consecutive 6-month periods spanning
1964 to 1965. Acceptable efficacy and no adverse effects up to 5 years following 27
neonatal exchange transfusions with ACD-A were noted by Kreuger.12' Falk, Lind-
blad, and Westman'la could find no histopathological evidence of adenine nephrotox-
icity in six of seven patients who underwent open-heart surgery in whom large quan-
tities of ACD-A were transfused. Westmanlzo could find no consistent evidence of
nephrotoxicity among 118 patients followed for 8 days after cardiac surgery during
which an average of 13 units of ACD-A were transfused. In Germany, Spielmann'''
and Seidl and S ~ i e l r n a n n reported
'~~ a favorable evaluation of red cell survival of
ACD-A-preserved blood stored for 3 to 6 weeks in 5 1 subjects. -
Widespread clinical use of ACD-A has occurred in Sweden; between 1964 and 1976
more than 2 million units were transfused. In 1976, A k e r b l ~ m "reported
~ that over
3200 units of CPD-A blood had been transfused and that this adenine blood preserv-
ative would soon be licensed in Sweden for general use. This formulation has one half
the adenine content of ACD-A (Table 1). Since 1976, CPD-A (0.25 mM) has gradually
replaced ACD-A in Sweden.'*O Clinical evaluation of the saline-adenine-glucose pre-
January I981 189
servative system (SAG, see discussion on optional additive systems below) is currently
underway in S ~ e d e n . ~ ‘ ~ By
. ~ *1970,
’ ACD-A with guanosine and inosine had been used
clinically in over 20,000 transfusions in Germany.144However, inosine is no longer
included in the blood preservatives used in Germany because of concern for potential
nephrotoxicity from the inosine metabolite uric acid.
At present, blood preservatives which contain adenine are routinely used in seven
European countries.’21 Blood bags sold by a major supplier in Sweden and Germany
contain predominantly adenine preservatives; in Sweden greaterthan 90% of such bags
contain CPD-A while in Germany, approximately 60% contain ACD-A. Of blood bags
sold by this supplier in France, Switzerland, Spain, Denmark, and Norway, 5 to 20%
contain adenine preservatives.
B. U.S. CPD-Adenine Clinical Trials

Efforts to obtain approval for clinical use of adenine-preserved blood in the U.S.
suffered a setback when renewed concern over DOA toxicity arose in 1969.8 However,
because of military interest in extending the storage life of blood, investigations contin-
ued. In 1974, the U.S. Army Blood Research Program undertook the job of coordi-
nating an adenine task force. The task force,* ultimately comprised of two blood bag
manufacturers and six blood preservation research laboratories, was convened specif-
ically to gather scientific information necessary to satisfy FDA requirements leading
to approval of an adenine blood preservative for general clinical use. A modification
of the standard CPD formulation was chosen which contained 17.3 mg of adenine per
63 mf of preservative solution (0.25 m M final whole blood concentration) and 125%
of the glucose content in standard CPD. This formulation, initially called CPD-11, was
later officially designated CPDA- 1. Although a protocol outlining general procedures
for the performance of Phase I, Phase 11, and Phase I11 clinical investigations was
devised, the FDA ultimately required the performance of only Phase I (autologous red
cell survival studies in normal volunteers). This development was largely due to the
recommendation^^^^ of the Bureau of Biologics (BOB) Panel on Review of Blood and
Blood Derivatives which convened o n October 1, 1976 in conjunction with a BoB-
sponsored Workshop on Adenine and Blood Preservation. 149 At the meeting, the task
force reported on 32 red cell survival studies performed by four cooperating labora-
tories.”’ Subsequently, the cooperating laboratories published the results of their
Phase I evaluation of 51 units of blood stored in CPDA-I for 28 to 35 days in the
July-August 1977 issue of Transfusi~n.’~ Prior to BOB approval in August 1978,’ 72
additional studies were performed. In all, 123 Phase I evaluations were performed by
members of the adenine task force, but results of the additional 72 studies have not
been published. For the purpose of this review, we summarize and comment upon
selected features of the entire U.S.CPDA-1 clinical trials data base.
Table 2 summarizes the results of the autologous SICr-tagged red cell 24-hr recovery
(Yo survival) studies of blood stored for 28 to 35 days as whole blood or red cell con-
centrates. Regression analysis of this data revealed no differences in Vo survival attrib-
* U.S. adenine task force was comprised as follows: Task force coordinators - T. F. Zuck, T. A. Bensin-
ger, C. C. Peck, A . M. Josephson, W. Davisson. M..Feldman. Manufacprers - Fenwal Laboratories,
1 Baxter Parkway, Deerfield, 111. and McGaw Laboratories, 2525 McGaw Avenue, Irvine, Calif. Coop-
erating investigators - T. A. Bensinger. C. C. Peck, T. F. Zuck, G . L. Moore, Division of Blood
Research, Letterman Army Institute of Research, San Francisco, Calif.; E. Beutler, R. K. Chillar. De-
partment of Medicine, City of Hope National Medical Center, Duarte, Calif.; S. V. Kevy, L. N. Button,
Children’s Hospital Medical Center, 300 Longwood Avenue, Boston, Mass.; P. R. McCurdy, Washing-
ton Regional Red Cross Blood Program, Washington, D.C.; K. Mayer, C. Zaroulis, Memorial Sloan-
Kettering Cancer Center, 1275 York Avenue, New York.; A. Orlina, Michael Reese Research Founda-
tion, Chicago, Ill.
190 C R C Critical Reviews in Clinical Laboratory Sciences
Table 2
24-HR "CR RED CELL RECOVERY
(0700). (U.S. CPDA-I CLINICAL TRIALS)
n Mcan+SD
35-day Whole blood 50 7 9 2 10

storage (Hct = 38 2 7)
Packed cells 57 71 2 16
(Hct = 7 4 i 7)
28-day Packed cells 16 76 & 13
storage (Hct = 7 4 2 4)
Blood stored in CPDA-I at 4OC
utable to manufacturer source of the bags. Although mean To survivals in 35-day

stored packed red cell units from three laboratories were similar (69.3 k 9.8, n = 8;
77.1 & 8.2, n = 15; and 74.0 k 8.0, n = i s ) , mean values from two laboratories were
relatively low (48.6 k 22.2, n = 4 and 45.4 & 16.0, n = 6) while the mean value of
one laboratory was relatively high (84.6 6.8, n = 6). The interlaboratory variability
was only partially explained by distribution of whole blood/packed cell units and stor-
age times across laboratories or by protocol deviations. * Variations in the performance
and analysis of the S'Cr-tagged red cell 070 survival studies also may have occurred. It
is surprising that 24-hr "Cr red cell recovery was chosen as the only measure of post-
transfusion cell survival.** With a little extra effort, additional clinically relevant
measures of cell survival could be derived from such studies. For example, estimates
of the fraction of cells removed from circulation rapidly after injection152and estimates
of the fraction and the mean life span of cells surviving greater than 24 hr may have
clinical relevance. The bulk of 070 survivals in whole blood stored for 35 days or packed
cells stored for 28 days exceeded 70%, the de facto standard'so for efficacy of red cell
preservation systems (Table 2 ) . While the mean value for 'To survival of packed cells
stored for 35 days exceeded 709'0,the results displayed a large SD; 19 of 57 units (33%)
fell below 70% survival. These results, supported by early studies,b have given impetus
to improve t h e CPD-adenine formulation.
Table 3 details six biochemical characteristics of blood stored in CPDA-I as reported
by task force laboratories. Variability in laboratory procedures performed in task force
laboratories may account for the size of SDs seen. However, the number of data points
used to obtain each mean is sufficiently large as to render the estimates reliable. One
possible exception is the plasma hemoglobin values where variation in sampling and
assay procedures are grounds to view the reported values with caution; they are likely
to be overestimates.
The interlaboratory differences discussed above in n o way invalidate the results of
this extensive effort.. An important benefit gained by the retrospective analysis, how-
ever, is an appreciation of the importance of the interlaboratory variability in collec-
* For example, one laboratory with a low mean olo survival reported on two units with hematocrits O f 89
and 91; the hematocrit required by the protocol was 75 3 5 .
** O n e cooperating laboratory routinely obtained a n estimate of rhe T,,-Cr. that is, the rime taken for the
concentration of "Cr in the circulating blood to fall to 50% of its initial value after correction for
physical decay. However, the International Committee on Standardization in Hematology has advised
against use of this index because of the difficulty in extracting information regarding mean life span.'"
Another laboratory obtained blood samples 56 to 7 6 days following injection of s'Cr-tagged CPDA-1
stored (35 days) red cells in four cases. These data led to estimates of mean life span of cells surviving
greater than 24 hr which were i n the normal range.
Table 3
SOME BIOCHEMICAL CHARACTERISTICS OF BLOOD STORED IN CPDA-1 (U.S. CPDA-1
CLINICAL TRIALS)
Days of storage (value 2 SD [number of units])
Measurement Unit type' 0 28 1T
Plasma hemoglobin (me%) WB 8.2 f 8.5 (49) - 46. I f 27.7 (49)

PC 4.0 f 9.3 (10) 194.4f87.9(11)
7.8 -t 8.4 (51) - 657.8f 816.8(51)
Plasma potassium (meq/f) WB 4.2 f 1.8 (48) - 27.3 f 4.6 (50)
PC 4.0* 1.4(10) 75.7f 11.4(16)
5 . 1 f6.2(51) - 78.5-t 21.5 ( 5 1 )
Plasma sodium (meq/f) WB 169.3 f 8.8 (48) - 155.0 9.2 (50)
PC 170.2 3.0(10)
* 122.2 * 18.4 (16)
169.4 f 15.5 (51) - 110.7-t21.5(57)
Blood glucose (mg%) WB 440.2 f 56.9 (50) - 229.3 ? 50.7 (50)
PC 456.4 f 58.0 (16) 8 4 . 0 2 33.6(16)
409.2 2 75.0 (52) 93.3*61.8(55)
Erythrocyte ATP (pmol/g Hb) WB 4.2 rt 0.7 ( 5 0 ) - 2.4 f 0.8 ( 5 0 )
PC 3.8 f 0.7 (16) 2.5 i 0 . 9 ( 1 6 )
4.2 -c 0.8 (57) - I .9 2 0.8 (57)
Erythrocyte 2.3-DPG (prnol/g Hb) WB 13.2 f 3.0 ( 5 0 ) - 0.7 -+ I .6 (50)
PC 13.5 2 1.4(11) 0.5?0.5(11)
12.7f 1.8(52) 0.4 0.4 (52)
. WB = whole blood; P C = packed red blood cells.

I92 CRC Critical Reviews in Clinical Laboratory Sciences
tive results of a cooperative clinical trial. Future cooperative clinical evaluations of

new blood preservation systems should incorporate strictly uniform methods and strat-
egies for assuring intra- and interlaboratory quality control.
The mean 070 survival of packed cells stored for 35 days reported here (71 2 16'7'0, n
= 57) differs from the 77 -+ 7.0% (n = 15) quoted in the BOB announcement of
approval of CPDA-I.' The latter represents a subset of the entire data base reported
here, which excluded protocol violations. Some of the I9 units which showed less than
70% survival were from units which were packed to hematocrits higher than 80. This
would indicate that it would be unwise to store blood with hematocrits higher than 80
for 35 days in CPDA-1.
At the present time, cooperative Phase I evaluations of an improved CPD-adenine
formulation, CPDA-2, are underway. CPDA-2 has twice the adenine content (34.6
mg/63 mf preservative solution) of CPDA-1 and 175% of the glucose content of stand-
ard CPD. This preservative formulation is intended t o ensure adequate 070 survival of
packed red cell units following 35-day storage. In vitro studies of CPDA-2 indicate
that A T P maintenance is superior to that in CPDA-14' and that there is no apparent
deleterious effect of the additional adenine on platelets. Cooperating laboratories
will determine several potentially relevant survival properties of red cells stored for 35
to 56 days as whole blood and packed cells. In addition, higher hematocrits (75 to 85)
of packed cell units and the effects of extended delays (up to 8 hr at room temperature)
prior to component preparation will be investigated.
C. Immunological and Biochemical Consequences of Prolonged Blood Storage

Prior to the introduction of CPDA-1, blood could be stored for only 21 days.
CPDA-1 allows blood to be stored for 35 days and thus extends by 14 days the in vitro
aging of all constituents. This section addresses the consequences of extended storage
on immunohematology, accumulation of intracellular compounds in plasma, microag-
gregate formation, and transmitted diseases.
Early in the development of blood preservation systems, it was observed that most
red cell antigens were stable during a limited storage period.lS3 Prolonged storage in
the frozen stateis4does not lead to a loss of antigenicity, except possibly PI antigen'"
and a pathological B-like antigen.Is5 Storage in C P D and ACD preservatives-does not
lead t o diminished antigenicity within 21 days, but C P D has been shown to exhibit a
minimal antigen loss after 28 Camp et al.lS7studied the effect of storage time
in ACD with adenine (0.48 mM) on red cell antigenicity as it pertains to crossmatching
procedures. Although this adenine formulation differed from CPDA-1, their observa-
tions may be used to speculate about antigenic effects of blood storage in CPD-adenine
formulations. There was no difference between fresh and preserved cells with respect
to titers of A, A,, B, A,B, and A,B antigens during 42-day storage. Using split ACD
and ACD-A bags, no differences were noted after 49 days of sorage in A,, A2, Rho,
Fy', M, N, S, and Xg" antigen titers. A one-tube dilutional difference.(tested in dupli-
cate) in B and K antigenic strengths was noted between the two preservatives after 49-
day storage; antigenic strength in the adenine-supplemented preservative was weaker.
The pointed out that the antisera used for Kell was weak. Evaluating anti-
gen-antibody reactions quantitatively by titration is insensitive and affords only a
crude estimate of the phenomena. Rosenfield et al. ,I5* using more-sensitive automated
techniques, evaluated red cell agglutination in low and normal ionic environments
from specimens stored for 70 days in ACD-A and CPD-2A. Loss of agglutinability
averaged 25, 50, and 75% at 4.1, 6.0. and 7.6 weeks of storage in both preservatives.
-
A, B, Rho, k, Jk', Fy', Fyb, and M antigens were tested. In the adenine supplemented
preservatives, 20 to 30% more agglutinability was seen from the 6th to the 10th week
compared to nonsupplemented preservatives (CPD and ACD). The diminished agglu-
January 1981 193
tination of stored cells did not appear to be due to a loss of antigenic sites; absorption
studies had shown no difference in the ability of stored and fresh cells to take up
specific antisera. These authorsLS8 concluded that the loss might be due to accumula-
tions of plasma protein on the surface of red cells. It appears that there are no signifi-
cant changes in antigenicity during storage for up to 35 days. In the future, if longer
storage periods prove feasible, the possibility of antigenic diminuation must be consid-
ered.
Accumulation of red cell metabolites in plasma during blood storage is usually re-
lated to the duration of storage.lS’ An additional 2 weeks of storage would be expected
to increase the metabolite concentrations in plasma, although perhaps not in a linear
fashion. Such an accumulation becomes important when blood is being given to pa-
tients who are unable to handle additional extracellular electrolyte and metabolite
loads. Plasma potassium levels, for example, rise during blood storage, and the
amount of potassium present in a unit of plasma of old stored blood (>14 days storage
time) is enough to cause concern when transfusing patients with hyperkalemia or im-
paired renal function.1S3However, concern about potassium load in patients with mas-
sive transfusion requirements may not be warranted because most of these patients are
hypokalemic after transfusion. l S 9 The mechanism for the latter phenomenon is not
fully understood, but acid-base disturbances and interstitial sequestration160appear to
be the most plausible explanations. An often implied159,161 mechanism of hypokalemia
seen after massive transfusion is the “potassium sponge” concept, whereby stored
donor cells depleted of intracellular potassium ions appear to re-establish the normal
Na-K gradient after reinfusion into the patient. The red cell potassium ion deficit,
according to this scheme, is met from the plasma pool of potassium. Studies from two
laboratories, however, d o not lend support for this c ~ n c e p t . ’ ~Valeri
~ . ‘ ~and
~ Huschi16’
have shown that the intracellular potassium concentration does not rise immediately
after transfusion; it may even fall initially before gradually returning to normal be-
tween 4 and 11 days. Crawford and M ~ l l i s o n calculated
’~~ that the rate of influx of
potassium ions into reinfused cells is no greater than that for fresh cells. I t therefore
appears that rapid shifts of potassium ions may not occur due t o an erythrocyte
“sponge’ ’ effect.
The rate of potassium ion appearance in plasma during storage has been reported
by several investigators.164-167 Michael et al. 164 have shown a linear accumulation over
a 3-week storage period, whereas o t h e r P ’ have shown a curvilinear accumulation pro-
file. The differences in kinetic characteristics may have been due to experimental tech-
niques since in the former study sampling was done o n undisturbed, cell-sedimented
plasma, whereas the latter study was performed with thorough mixing before sam-
pling. The plasma potassium concentrations may be artifactually affected by sampling
methods which promote hemolysis or nonhomogenous mixing. Michael et a1.16‘ re-
ported 21-day storage values of about 12 meq/l for whole blood and 40 meq/l for
packed cells. report values a s high as 21 meq/l after 21 days of whole blood
storage. The relationship between plasma storage volume and red cell mass can be
invoked to explain some of the differences reported in the literature. This relationship
is shown in Figure 6 which depicts a rise in plasma potassium concentration following
35-day storage which is proportional t o the hematocrit.222Althpugh the plasma potas-
sium concentration rises in packed cell units, the total potassium load per bag remains
less than that of whole blood stored for a comparable storage period.’64
The effect of adenine in CPD and ACD on plasma potassium concentrations in
blood stored for prolonged periods has been studied by several group^.'^^^^ 1 6 7 . 1 6 8 In
blood stored for 28 to 42 days, an increase of 5 to 25% in potassium concentration
was observed compared to 21-day levels, depending on hematocrit and period of stor-
age. The presence of adenine does not appear to affect plasma potassium levels4’
194 C R C Critical Reviews in Clinical Laboratory Sciences
4- +
+
+
30 40 50 60 70 90
Homatocrit
FIGURE 6. Plasma potassium (meq/f) in relation to hematocrit of blood stored (4°C) for 35 days in
CPDA-I (n = 1 1 ) . (Courtesy of Bolin, R . B.. unpublished analysis of U.S. CPDA-I clinical trials data
base, Letterman Army Institute of Research, San Francisco.)
(Figure 7)."' The failure of adenine to influence potassium shifts during storage adds
credibility to the hypothesis that cation changes are due to temperature-dependent ac-
tivity of ATPase rather than the actual A T P content of the red cell'69 since higher
ATP levels are maintained with adenine supplementation. From the foregoing data,
the clinical situations which would necessitate concern about infused potassium load
(e.g., neonatal infusions, renal disease, massive muscle injury) must still be met with
appropriate hemotherapy (relatively fresh stored blood, washed red cells, etc.), but in
most clinical situations, the additional potassium load seen in 3 to 5 weeks of storage
would not be excessive. Plasma sodium levels remain relatively constant throughout
30 days of and are not influenced by the presence of adenine (0.5 mjM).42
Ammonia accumulation in plasma during storage could be undesirable when trans-
fusing blood into patients with compromised liver Bailey and B o v ~re-' ~ ~
ported blood ammonia levels obtained from ten units of whole blood stored in C P D
for 28 days. The initial ammonia levels are questionable because of possible difficulties
with the ammonia a ~ ~ a yInitially,
. ~ ~ mean
~ . ~blood
~ ~ammonia concentration was 282
pg/dl, which is almost three times higher than the upper limit of normal for the am-
monia assay technique The elevated levels may have been due to ammonia
produced during sample processing delays. The authors did not find any free ammonia
in their CPD solution. After 24 hr of storage, the ammonia concentration had dropped
to a mean of 178 pg/dl, after which time it rose in a somewhat linear fashion to a
mean concentration of 705 pg/dP at 28 days. Assuming that the reported levels of
ammonia are accurate and that ammonia is distributed solely within the intravascular
space, the blood ammonia level in a 70-kg man would be expected to rise by 60 pg/dP
with the transfusion of one unit of 28-day old whole blood. Although blood ammonia
January 1981 195
20$/ .______.-..
&CD
- - -CPDA-1
-CPD
- .
0 7 14 21 28 35 42
TIME (DAYS)
FIGURE 7. Plasma potassium concentrations (mean 5 SD. n = 6 ) during storage (4’C) of packed red
cells (mean Hct = 69.3%) in three anticoagulants: ACD, CPD, and CPDA-I. (Courtesy of Neher, J . and
Bensinger, T. A., unpublished data, Letterman Army Institute of Research, San Francisco.)
level, particularly in venous samples, may not be correlated with clinical hepatic
coma,171the added ammonia can be detrimental in a marginally compensated host.
Figure 8 shows the plasma ammonia concentration in blood stored in ACD, CPD,
and CPDA-I for 42 days.’” The initial values are much lower than those of Bailey
and Bove, 16’ but these may represent differences in ammonia assay or sample process-
ing technique. After 35-day storage, a 70-kg man would receive about 3500 ,ug of am-
monia per unit or about 3 1 O7o more ammonia than he would receive from a comparable
unit stored 21 days. On the other hand, the ammonia load from CPDA-1 is n o worse
than with C P D any time during 21-day storage. Blood stored in ACD appears to pro-
duce less ammonia than blood stored in C P D preservatives. These data suggest that
blood stored beyond 21 days is capable of presenting a significant ammonia load t o
patients with compromised liver function. As with conventional systems, careful con-
sideration must be given to these patients’ hemotherapy needs, particularly when con-
templating the use of stored blood. For hemotherapy in a patient with severe hepatic
insufficiency, relatively fresh (less than 5 days old) units are preferred. I f older blood
must be used, removal of plasma from whole blood and, preferably, washed red cells
may be recommended.
Spontaneous hemolysis occurs during blood storage and is related to age of the
blood. although the presence of free hemoglobin in the plasma of blood stored in
conventional preservatives (CPD, ACD) has not correlated well with in vivo red Cell
s ~ r v i v a b i l i t y In
. ~ ~an~ adenine preservative system, a correlation has been r e p ~ r t e d . ’ ’ ~
The mechanism is not clear whereby hemolysis occurs during blood storage. Although
red cells lose apparent V C I ~ U ~shape, ~ , ‘ ~and
’ deformability during storage, such
1000 T T
OT t
0 7 14 21 28 35 42
TIME (DAYS)
FIGURE 8. Plasma ammonia concentrations (mean f SD, n = 6) during storage of packed red cells in
three anticoagulants: ACD, CPD, and CPDA-1 (Courtesy of Neher, J . and Bensinger, T. A . , unpublished
data, Letterman Army Institute of Research, San Francisco.)
changes are probably signs of membrane defects which may contribute to lysis. Mem-
brane storage lesions have been described and crudely measured.’” Membrane endo-
cytosis is altered during storage and is reversible with restoration of A T P level^."^
This suggests that energy-dependent mechanisms such as membrane phosphorylation
or protein kinase activity are affected. Such defects could alter the lipid-protein com-
ponents and thus modify the solid material elasticity of intact red cells.’74 A second
reversible mechanism of in vitro hemolysis is based on an interesting observation in
artificial media systems made by Hogman et al.lrs They showed that red cells sus-
pended in saline-adenine-glucose (SAG) hemolyze to a greater degree than blood stored
in a protein medium. These author^'^' observed that hemolysis could be reduced by
removing the buffy coat or by supplementing the preservative with enzyme inhibitors.
They postulate that during storage white cells liberate a chymotrypsin-like enzyme with
potent hemolytic effects. This enzyme is inhibited by plasma inhibitors not present in
the artificial media. The concept of white blood cell influence in stored blood is not
new. Ambrus et al.42showed that packed red cells stored as buffy coat-poor units have
greater 24-hr postinfusion survival than comparable units with buffy coats. A contri-
bution of white cell proteolytic action to in vitro. hemolysis in stqred blood is possible
and should be investigated. A third mechanism whereby hemolysis may occur in vitro
is from physical stresses. I t is well known that physical trauma and heat cause lysis
and that stored red cells are particularly susceptible to these Aspiration of
red cells through small needles or under pressure can induce hemolysis. Although prob-
ably not responsible for all of the hemolysis observed with storage, mechanical injury
may explain part of the differences observed among laboratories and the wide range
of values reported in the U.S. CPDA-1 cooperative studies.
January 1981 197
Several reports of plasma hemoglobin levels during prolonged storage using preserv-
atives with and without adenine have been published.1s 4 2 I Z 2 1 6 7 1 7 6 These studies are
difficult to compare since some authors1” report free hemoglobin as percent lysis and
others as free hemoglobin concentration. I s 1 6 7 176 In addition, little attention is paid, at
least in print, t o the problem of appropriate hemoglobin assay methodology and the
accuracy and precision of results. For example, loss of accuracy and precision has been
noted when the benzidine hemoglobin assay, a method which works well at low hemo-
globin concentration (3 to 25 mg/dP), is used to measure the higher hemoglobin con-
centrations in plasma of stored blood.’77Since in these studies1’ 4 2 1 2 2 1 6 7 176 methods of
plasma hemoglobin determination were either not reported o r were benzidine methods,
the validity and accuracy of reported values cannot be assessed. Ambrus et al.4*did
not note any differences between plasma hemoglobin levels in ACD and ACD-A. Mes-
seter et al.’76couldfind no difference between CPD-A (0.25 mM/P) and ACD-A.
The amount of free hemoglobin in a unit of transfused blood is probably not of
clinical significance in most situations. A k e r b l ~ m reported
’~~ that mean serum hapto-
globin concentrations in 10 recipients decreased 3.5% 1 hr after transfusion of blood
stored for 29 to 35 days in ACD or ACD-A. Assuming milligram for milligram binding
of haptoglobin (- 100 mg/dl norrnalty) with hemoglobin, approximately 90 mg/dP of
free hemoglobin can be calculated to have come from infusion of free hemoglobin in
each unit. This rough estimate is higher than that reported by Zuck et aI.15 (plasma
hernoglobin = 50 mg/dl) for CPDA-1, but lower than that reported by Messeter et
al.176(1 10 mg/dl) after 28-day CPD-A storage. Assuming a free hemoglobin concen-
tration of 90 mg/dP in each transfused unit, a 70-kg man given 10 units of whole blood
would consequently have a plasma hemoglobin level of about 80 mg/dP. Since this
load of hemoglobin would be bound entirely to haptoglobin, no free plasma hernoglo-
bin would be detectable. A theoretical clinical problem could be postulated if the rare
ahaptoglobinemic individual or phenotypic H p 2-2 haptoglobin individual with low
basal haptoglobin levels were t o receive blood stored containing free hemoglobin. In
these cases, small concentrations of free plasma hemoglobin could result in a pinkish
in the patients’ plasma. Trace hemoglobinuria might also be detected if free
hemoglobin exceeded 25 mg/dl. A confusing clinical picture could result which might
suggest intravascular hemolysis. This example points out the clinical problem of inter-
preting posttransfusion plasma haptoglobin without pretransfusion levels for compar-
ison. Massive transfusions of greater than one blood volume could result in free plasma
hemoglobin due to a dilutional loss of haptoglobin as well as infusion of exogenous
hemoglobin from stored blood. Neonates, who normally d o not have haptoglobin,
could present a similar picture following transfusion of smaller volumes of blood.
However, neonates d o not ordinarily receive older blood because of associated prob-
lems (potassium, ammonia, and acid load).
Acid-base alterations due to acid load of stored blood have not been considered a
problem in recent years since blood gas analysis is widely available and since CPD
preserved blood is used instead of ACD blood.’6’ Furthermore, the relationship of
infused acid to the buffering capacity of the human body is complex and is largely
determined by perfusion and endogenous acid production. The acid in stored blood is
primarily lactic and citric acid, and both can be readily metabolized with adequate
p e r f u ~ i o n . ’Nevertheless,
~~ there is that the addition’al hydrogen ion load
contained in blood stored for prolonged periods may be detrimental in certain situa-
tions, especially with elderly patients. There is n o difference in prestorage p H of blood
harvested in C P D or CPD-adenine (pH = 7.0 to 7.2 at 37°C). Bailey and Bovel6’
reported a pH of 6.9 in all 10 units of blood stored in C P D for 28 days. Considering
the lack of variability and the high pH values, one would have to assume their mea-
surements were insensitive and that samples were measured at a nonphysiological tem-
perature. Using p H measurements at 37"C, Moore et aL4' reported that there was no
difference between blood stored in C P D and C P D with three concentrations of adenine
and two concentrations of glucose throughout 42 days of storage. pH after 28-day
storage approached 6.6 in all preservatives.2Z' Messeter et and Dawson et a1.178
showed pH values similar to those of Moore et al.41 in blood stored for 28 days in
various CPD-adenine formulations. According to the latter two groups, pH became
asymptotic by 28 days. Messeter et al.,176however, pointed out that by 28 days of
storage, CPD-adenine preservatives show no advantage over ACD-adenine insofar as
pH is concerned. When blood stored for longer than 28 days is used in massive trans-
fusion situations, particularly those involving elderly patients, the monitoring of acid-
base status may be required. However, the magnitude of this problem has yet to be
adequately defined.
The effect of adenine and prolonged blood storage on microaggregate formation is
of possible concern; however, the exact role of infused microaggregates in subsequent
organ damage has not been established.'79 Although no studies of microaggregate for-
mation in adenine-preserved blood have been published, microaggregate formation has
been studied in blood preserved in C P D and ACD for 21 days.'s0.'81 The formation of
microaggregates is time dependent, but the bulk of debris formation as measured by
screen filtration pressure and microaggregate protein occurs by 7 days; additional in-
formation of microaggregates, especially between the second and third weeks, appears
minimal.182This suggests that storage for as long as 5 weeks would not appreciably
increase microaggregate debris; however, studies of microaggregate formation in ex-
tended storage systems containing adenine are needed to test this postulate.
Effects on transfusion-transmitted disease in blood stored for a prolonged period
have not been reported. However, there is n o reason to assume that the adenine pre-
servatives will alter the incidence of hepatitis, cytomegalovirus infection, o r other mis-
cellaneous infectious disease.
Even though minor questions about the efficacy of adenine-supplemented preserva-
tives and the effects of prolonged storage may be entertained, there are considerable
data supporting the extension of storage of adenine-supplemented blood to 35 days
Clinical situations exist in which clinicians must be aware of the hazards of transfu-
sions, but these are time-of-storage related factors rather than adenine-related ones.
The use of blood stored beyond 21 days should not pose insurmountable problems at
the clinical level since only patients with limited tolerance to metabolites leaked from
red cells in conventionally stored blood will require special hemotherapy consideration
with the newer systems. Patients receiving large volumes of 35-day stored blood will
have to be monitored for electrolyte and acid-base changes. In most cases, there should
be n o additional problems due to toxic metabolites in transfused blood. The recom-
mendation to monitor electrolyte and acid base status reflects good hemotherapy prac-
tice more than concern about the use of a new preservative.
D. Impact of Adenine-Preserved Blood Banking Logistics

The storage of adenine in a high glucose concentration may result in an instability
of adenine. A U.S. manufacturer currently limits the storage time of blood bags con-
taining CPDA-1 to 15 months after manufacture in contrast to ?6 months for standard
CPD bags. Such a restriction may adversely influence the blood bag inventories which
blood banks have been accustomed to with the 36-month product. The costs of man-
ufacture and supply may be higher'for CPDA-I due to the shorter bag storage time.
These problems could impede further development of improved adenine preservatives
(e.g., CPDA-2) unless improved preservative stabilization can be enhanced.
There is little doubt that an additional 14 days of storage life will have a significant
January 1981 199
-
0
0
-
-
X
nl
.-
C
a
0
L
0
\ \
n
E
a
- - - 21 day shelf-life
z 35 doy shelf-life
't0 4 12 16 20 24 28 32 36
TIME (DAYS)
FIGURE 9. Simulated distribution of stored blood units in a regional blood center. Values given are the
mean number of units (vertical axis) 2 SD which are of a given storage age (horizontal axis). The mean of
four simulations representing 1 year is depicted. (Courtesy of Bolin, R. B . , Gaddis, R . , and Szurek, J . L.,
unpublished data, Letterman Army Institute of Research, San Francisco.)
impact on the logistics of blood supply. The 15-year experience of use of adenine-
supplemented blood in Sweden is claimed to have decreased blood wastage through
outdating. Stochastic computer simulation models have forecasted reduced outdat-
ing as a result of extending storage time beyond 21 day^.'^^-'^^ A reduction of one half
to two thirds in outdating, corresponding to a I-week increase in available inventory
for a time regional blood bank system, seems possible by such estimate^.'^^-^^^ How-
ever, the introduction of adenine-supplemented blood into blood banking in the U.S.
may not result in drastic changes in the outdating rate. Blood wastage has received
national attention since 1972'R6.187 when a blood resource study estimated that one unit
out of every four drawn was destined to be outdated. Current management tech-
niques188-190 which are aimed at more efficient blood distribution can reduce wastage
within the confines of 21-day shelf life. Since most blood in the U.S. is supplied on a
regional distribution basis, rlecycIing of older blood from low-probability-of-use to
high-probability-of-use hospitals can have a significant impact on a national blood
supply. The nationwide wastage figure of 15% reported by the American Blood Com-
mission for 1978 may reflect improved intraregional blood dist~ibution.'~' The use of
the nationwide wastage figure as an index of the overall impact of CPDA-1 must be
interpreted in the light of current management strategies. For example, Figure 9 depicts
the storage distribution of blood managed by a rurally oriented regional blood center.
The data are based on actual operating figures and management decisions for a 3-week
period, expanded by computer simulation to four 90-day periods.ZZsThe data represent
the mean number o f units arid SD according to storage age for these four periods.
Table 4
COMPUTED SIMULATED DATA ON A BLOOD
BANK OPERATION
Storage life
21-day 35-day Probability'
Average daily 431.2 r 16.0 455.9 -c 11.7 0.047

stock size
(units)
Average daily 6.8 c 0.9 6.4 f 1.4 0.640
unable-to-fill
units
Daily wastage 2.9 r 0.7 0.02 2 0.02 0,005
(070)
Daily out of re- 22.4 2 3.7 29.2 f 2.5 0.051
gion shipments
(units)
a
Probability that the null hypothesis is true.
Maintaining identical management decisions for 21- and 35-day blood storage life re-
sults in little difference in age distribution throughout the region. The peak at 10 days
reflects redistribution. In the 35-day storage simulations, only 1.43% of all blood avail-
able at any time was older than 21 days. Since the only units not accounted for by this
model are the transfused or outdated blood, the limited availability of older blood
would not be expected t o influence the outdate rate greatly.
Although an advantage of longer blood storage life might be to decrease blood was-
tage, there are other important considerations. Simon9 has suggested t h a t extending
blood storage life may have beneficial effects on the economy, efficiency, and flexibil-
ity of blood supply. The additional storage time could increase blood inventories, re-
duce transfers, improve service to remote areas, and improve collection strategies. Bro-
dheim and HirschI9* predicted an improvement in blood bank service in a large blood
service region with an urban orientation as a result of extended storage life. They
argued that time of redistribution can be extended from about the 8th day to the 10th
to 17th day without affecting wastage, resulting in reductions in costs and blood short-
age rates. Although the data were gathered in a rural regional blood service, Figure 9
illustrates their point. If the day of redistribution (which for this geographically large
region is 10 days rather than 8 days) is shifted to a later storage age, for example to
the 17th day, then the number of units being transported for redistribution purposes
could be reduced by as much as 2% times. The cost reduction in this region could be
dramatic since current transportation expenses are about $1.40 per unit of blood.2z6
Advantages in blood supply management require different decision-making strate-
. ~ 4 displays some of the data
gies to provide the better service outlined by S i m ~ nTable
obtained from the rural regional simulation model. "' Although the age distribution of
blood is not significantly altered, there are changes in the distribution statistics between
21- and 35-day-old blood. There is about a 5 % increase in available stock (uncross-
matched stock), a considerable drop in the nonhospital wastage,* and about 30% more
* Nonhospital wastage is the amount of blood wasted in relation to total drawn by a region as a whole
because of an inability to distribute or redistribute before the units outdate. As such, i t is an index of
inventory management efficiency."' The majority of outdated units in the region under study are not
accounted for by this index.
January 1981 20 1
units available for out-of-region shipment. The simulations d o not show the ability to
lower the number of unable-to-fill orders. However, “unable-to-fill” is defined for
this model as any order unfilled as specifically ordered by the hospital, even though
the order may have been modified with the approval o f the hospital (e.g., packed cells
substituted for whole blood). These differences were due entirely to blood storage du-
ration of the same decision-making processes and were independent of management
decision-making processes.
Changes in decision-making policy can be made to improve a blood supply system
based o n extended blood storage technology. For example, the decision to ship blood
out of a region is based on the stock size, age of blood in stock, and available market.
Should increased age of blood prove t o be a negative factor for out-of-region market-
ing, then a decision not to increase this market would result in approximately seven
units a day more in stock. I f intraregional needs are already met, this decision could
result in an increase rather than a decrease in nonhospital wastage. A decision to reduce
the daily collection of blood by 8 % would prevent the wastage of those additional
seven units.
With a longer shelf life, the potential for improved blood service is now available.
The ability to provide improved service depends upon the acceptance of the new pre-
servative (e.g., CPDA-I) by the medical community, good inventory management by
hospital blood banks, and an awareness by regional blood services of the need to re-
align management decision policies to reflect the change in the perishability of their
product. At the same time, decision makers cannot rely on the additional longevity as
a management tool, but rather the longevity relies on sound decisions.
V. EXPERIMENTAL BLOOD PRESERVATION SYSTEMS

CONTAINING ADENINE
A. Optional Additive Systems

Knowledge gained from experience with adenine-supplemented blood preservatives
logically leads to the next major advance in blood storage technology, optional additive
systems (OAS).’93In OAS, blood is collected into a standard anticoagulant such as
CPD or CPDA-1 followed by separation into components. After component separa-
tion, the user has the option of adding special preservative chemical(s) to each cellular
component. The advantages of this approach are severalfold:
1. A unique preservative for each individual blood component allows greater flexi-
bility in the design of the solution for optimal results
2. Testing of a given system for FDA approval would be limited to that blood com-
ponent(s) for which thr: OAS system was developed
3. Hematocrit effects would be eliminated (cf. placing additive in anticoagulant),
allowing for maximal harvesting of desired components such as plasma
The advantages of component-specific preservation of red cells became apparent

during the development of CPDA-1. The formulation for CPDA-I provides 17.3 mg
of adenine per 500 mP of whole blood. At a hematocrit of 40, 17.3 mg of adenine is
available to about 200 m l o f red cells, yielding an effective reservoir of 87 pg adenine
per m l of red cells. This quantity of adenine is sufficient t o maintain red cell ATP
concentrations in whole blood during 35 days of storage such that subsequent 24-hr
”Cr red cell recoveries averaged 79 +- 10% (Table 2). However, when the red cells are
stored as packed cells, the lyortion of the adenine remaining in the plasma folIowing
initial equilibration (Figure 2) is removed during component separation. The exact
amount depends upon the hematocrit to which the cells are packed, even though the
mean red cell volume remains at a constant 200 m l . At a hematocrit of 70, plasma
removal results in a 33% loss of the adenine originally available to the red cell mass,
and packing to a hematocrit of 95 results in a 45% loss. The latter is equivalent to a
reduction in the effective reservoir to 48 pg/ml red cells. The same argument can be
made for glucose, further demonstrating that storage as packed cells results in nutri-
tional deprivation of the red cells when compared to whole blood storage. The lower
mean 24-hr red cell 5’Cr recoveries of 71 k 16% (Table 2 ) obtained with CPDA-1
packed cells is also evidence of this nutritional deprivation. For optimum red cell stor-
age, it would be desirable to determine and maintain an effective reservoir of adenine
and glucose per unit of red cell mass. This situation would be more closely approached
if the adenine and glucose were added to the red cells after packing; moreover, this
would allow for the maximum harvesting of plasma without worry of compromising
red cell quality.
In the CPDA-1 cooperative studies, it was a slow and tedious process to demonstrate
clinically that adenine had no deleterious effects on platelet concentrate storageability.
This problem would be entirely eliminated if component-specific blood storage systems
were incorporated into the blood banking system. It might also make improvements
in blood storage systems more economical and quicker to develop since testing and
evaluation would be simplified.
Preliminary attempts at the component-specific approach to improved blood storage
may be illustrated by several recent proposals for improved methods for packed red
cell storage. In 1975, Zuck and B e n ~ i n g e rdiscussed
’~~ the advantages of sterile docking
devices which would permit the addition of a preservative solution to packed red cells
after component preparation. They proposed the term “Optional Additive System”
or OAS for the system since the blood bank might have the option of when or if to
use the additive depending on the age of the unit and the specific demands on the
blood bank. Some red cell additives such as adenine and glucose may be added up to
14 days after the red cells are d r a ~ n ~and~ ~still
’ ~ be
’ efficacious during long-term stor-
age. Therefore, if the units were used within 2 weeks, the OAS solution would not be
added, hence eliminating the risk of any adverse effects of the additives to recipients.
Units kept longer than 2 weeks would receive the OAS solution for long-term mainte-
nance of red cells. Another strategy using the OAS concept centers on the addition of
2.3-DPG-maintaining compounds, such as dihydroxyacetone, which are stable in SO-
lution by themselves, but unstable if incorporated into C P D at the time of bag manu-
facture. 193.194 Zuck and BensingerL9’were considering the use of sterile docking devices;
alternately the OAS solution concept could be used by inclusion in a small satellite
bag containing the additives which would be directly attached to the primary bag. This
might not add significantly to the cost of a multiple bag system and could be cost-
competitive with sterile docking devices. Strauss, Meurer, and D e k o ~ s k i studied’~~
packed red cells which were resuspended and stored in a solution of xylitol, sorbitol,
adenine, guanosine, bicarbonate, and phosphate. They demonstrated that after 42-day
storage in this medium, red cells showed higher A T P and 2,3-DPG than cells stored
in CPD, retaining 73% of their A T P and 20% of their 2,3-DPG by day 42. These cells
were made “buffy coat poor” and packed to hematocrit z 5 5 . On day 42, the mean
24-hr 51Crred cell recovery was 77 & 7% (n = 5 ) :
Hogman et al. 146.147 have developed an optional additive system containing saline,
adenine, and glucose which they call SAG. In their system, red cells are drawn in C P D
packed to a hematocrit of 90, then they are mixed with 80 to 100 m l of SAG solution
and subsequently stored for 35 days at 4°C. The SAG solution yields red cells which
have 70% of their initial A T P concentration on day 35 and which were associated with
a 24-hr ”Cr recovery of 83 k 6.8Vo. The use of 80 m l of SAG lowered the red cell
January 1981 203
suspension viscosity to a level (Hct 2 60) which permitted transfusion without further
dilution. Hemolysis was higher in SAG than in packed cells and was attributed to
proteolytic activity from damaged leukocytes. The hernolysis could be reduced by re-
moval of buffy coat at the beginning of storage or by the addition of the protease
ir.!!ibitor, E-aminocaproic acid, to the red cells. stated that SAG appeared
to be an effective way t o harvest components, but that a requirement for whole blood
remains and that mixtures of packed red cells plus commercial plasma are not a rea-
sonable substitute. Schmidt 1 9 6 further argued that one half of the blood transfused
should be whole blood and urged blood centers to keep component preparation and
the use of SAG-like systems in perspective.
Moore et al. 19' have evaluated an OAS solution containing glucose, adenine, and
dihydroxyacetone. Addition of this solution to packed red cells (Hct = 75) drawn in
CPD showed significant improvement in maintenance of both the red cell ATP and
2,3-DPG concentrations during 42 days o f storage. This OAS solution produced ATP
concentrations which were 5 0 t o 60% of initial values o n day 42 and 2,3-DPG concen-
trations which were 60% of normal on day 21 and 20% of normal on day 42. The
solution may be added to the packed cells on the day of harvesting o r up to 7 days
later with no difference in results.
Bensinger, Chillar, and B e ~ t l e r ' ~ ' have
. ' ~ ~proposed the storage of red cells in a me-
dium called BAGPM, which contains bicarbonate, adenine, glucose, phosphate, and
mannitol. In this system, blood is drawn into ACD and the plasma components are
harvested as usual. Then 200 m l of the BAGPM solution is added to the concentrated
red cells in the primary bag; this bag also contains a silastic block impregnated with
calcium hydroxide. During blood storage, the metabolically generated acid reacts with
the bicarbonate to generate CO, which is absorbed by the C a ( 0 H b . In this manner,
pH is maintained at or above initial values throughout 42 days of storage. Use of the
BAGPM system resulted in 2!,3-DPG maintenance (78% on day 42) and marginal ATP
maintenance (44% on day ,,).I9' This system also exhibited reduced microaggregate
formation when compared t o that in conventional A C D o r C P D storage media. 19'
The consideration of specific preservative solutions for red cell storage is in its in-
fancy. There is promise that some formulation, probably containing glucose, adenine,
and other nutrients, will not only produce optimally preserved red cells, but also pro-
vide cells with improved oxygen delivery capabilities as defined by prolonged mainte-
nance of high 2,3-DPG concentrations. This general approach is also applicable to
platelets.
B. Optimal Blood Preservation Systems

Development of new blood preservation systems has proceeded toward the attain-
ment of certain desirable goals. Early systems were aimed at anticoagulation and pres-
ervation of viable red cells for a minimum storage time (e.g., citrated blood).199There-
after, systems were developed which extended storage time of viable red cells to 21
days and beyond (e.g., ACD and C P D and later ACD-A, CPD-A, CPDA-I, and
CPDA-2). 1s~12s.200~201Recently, maintenance of near-normal oxygen-transport proper-
ties as long as possible during storage has been added to the list of desirable goals.2o2
Although a wide variety of candidate preservative constituents has been investigated,
experimenters eventually agreed upon the several basic ingrediehts (including adenine)
resident in currently availablle blood preservatives. During the period which spans the
developmental work on ACI) and OAS (1940 to 1980). the growing body of knowledge
about red cell metabolism enabled scientists t o manipulate preservative constituents
which influence key erythrocytic metabolites affecting viability and function (e.g.,
ATP' and 2,3-DPGz11.204). The exact choice of ingredients as well as their concentra-
tions in the preservative solution hence appears to have resulted from systematic bio-
chemical investigations coupled with in vivo cell survival
204 CRC Critical Reviews in CljnicaJ LaboraforySciences
Developers of blood preservatives have claimed206-208 or implied 1 2 5 . 1 9 7 - 2 0 1 that their

choice of preservative constituents and concentrations yielded optimal preservatives;
however, it is not at all clear that this is the case. The uncertainty in this regard arises
from the lack of evidence that any particular preservative is truly superior to other
possible formulations. It would appear that systematic investigations of the effects of
temperature, pH, phosphate ion, and storage time on red cell ATP and 2,3-DPG, such
as those of Beutler and DuronZo9and Dawson and his group,210.211 satisfy the question
of optimality. However, in these experiments, only the factor of interest was varied,
while all others were held constant. This experimental approach can be extremely val-
uable for identifying the independent effects of the factor of interest at particular levels
of the others. However, unless all factor effects are independent of each other, choice
of preservative specifications based on such one-factor-at-a-time type studies may not
be optimal. Interactive effects of preservative constituents almost surely exist and un-
less they are studied, the exact combination of preservative constituents and their con-
centrations which yields optimal outcome of storage remains unknown.
Any uncertainty regarding the optimality of currently available blood preservatives
does not detract from their usefulness. However, development of new blood preserva-
tion systems may take advantage of modern methods of experimental design which
specifically aim towards discovery of optimal systems. Members of this class of exper-
imental designs, sometimes called response surface methods,212include two approaches
which are particularly well suited to blood preservation research and development: the
simplex technique and the factorial experiment. The simplex technique uses the results
of recent experiments to define new experimental conditions which ultimately lead to
optimal r e s ~ l t s . ~Only ~ ’ ~instance of the use of this technique in blood research
~ ~ . one
has been reported: Reiss and Katzzls used the simplex technique to define conditions
of centrifugation (speed and duration) which result in optimal (maximal) platelet yield
during separation from freshly donated blood. The simplex technique can be used most
effectively when experiments can conveniently be performed in a serial fashion and do
not involve a long period of time for each experiment. For instance, the simplex tech-
nique may work well in guiding platelet preservation studies in which individual exper-
iments can be performed within a week or two. In contrast, red cell preservation studies
typically require in excess of 3 to 6 weeks to perform. In this instance, it is more
efficient to vary several preservation conditions simultaneously in one large experiment
than to perform individual experiments in serial fashion. The response surface tech-
nique applicable here is the factorial experiment.*” Using this approach, the investi-
gator can perform a blood preservation experiment involving several concentrations
of each of the preservative constituents. The results could be analyzed by using poly-
nomial regression techniques and calculus to locate optimal conditions. Fractional fac-
torial designs are available to reduce the amount of experimentation necessary to ob-
tain more limited but yet useful results.”’ Application of this approach to the
development of a CPDA-I /OAS containing dihydroxyacetone which yields optimal
red cell 2,3-DPG and ATP during 42-day storage has been reported.’I6 An instructive
example of the use of fractional factorial experimentation in the development of an
optimal drug formulation was reported by Fonner, Buck. and Banner.218
January 1981 205
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Adenine Blood Preservation

Article in Critical Reviews in Clinical Laboratory Sciences · February 1981

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ADENINE IN BLOOD PRESERVATION

U.S.Food and Drug Administration (FDA) approval of the anticoagulant-preserv-

A. Transport of Adenine into Red Cells

as quantitatively and mechanistically as that of carbohydrate transport, but several

B. Metabolism of Adenine by Red Cells

HGPR is hypoxanthine guanine phosphoribosyl, APR is adenine phosphoribosyl.

PACKED CELLS HCT=70

FlGURE 3. Red cell 2.3-diphosphoglycerate (2.3-DPG) during 42-day storage (4’C)

Two recent ~ t u d i e s ~ ' concerning

tion of adenosine to inosine and its conversion to hypoxanthine (instead of conversion

C. Transport of Adenine into Platelets

D. Metabolism of Adenine by Platelets

Extracellular Pool N o n g r a n u l a r Pool Granular Pool

FIGURE 4. Schematic of adenine transport, compartmentalization, and metabolism in human platelets.

here.^'.^^ Platelets, like erythrocytes, are unable to synthesize purine nucleotides de

E. Subcellular Distribution and Metabolic Function of Adenine Nucleotides

Conflicting reports may be explained by the difficulty of separating adhesion from

F. Biochemical Effects of Adenine on Platelets under Storage Conditions

A. Metabolism and Pharmacokinetics of Free Adenine in Man

AEC = (ATP + %ADP)/(ATP + ADP +- AMP)

DISPOSITION OF INFUSED ADENINE IN M A N

patients suffering from hemolytic disease of the newborn. Kreugern9demonstrated in

B. Recipient Toxicity from Adenine in Blood Products

C. Effects of Adenine on Components of Stored Blood

inhibition of osmotic recovery could be induced directly in a dose-dependent fashion

D. Effects of Adenine on Plasticizer Leaching and Metabolism

IV. CLINICAL USE OF ADENINE-PRESERVED BLOOD

A. Use of Adenine-Preserved Blood in Europe

B. U.S. CPD-Adenine Clinical Trials

35-day Whole blood 50 7 9 2 10

Blood stored in CPDA-I at 4OC

utable to manufacturer source of the bags. Although mean To survivals in 35-day

Days of storage (value 2 SD [number of units])

Measurement Unit type' 0 28 1T

Plasma hemoglobin (me%) WB 8.2 f 8.5 (49) - 46. I f 27.7 (49)

. WB = whole blood; P C = packed red blood cells.

tive results of a cooperative clinical trial. Future cooperative clinical evaluations of

C. Immunological and Biochemical Consequences of Prolonged Blood Storage

D. Impact of Adenine-Preserved Blood Banking Logistics

21-day 35-day Probability'

Average daily 431.2 r 16.0 455.9 -c 11.7 0.047

V. EXPERIMENTAL BLOOD PRESERVATION SYSTEMS

A. Optional Additive Systems

The advantages of component-specific preservation of red cells became apparent

B. Optimal Blood Preservation Systems

Developers of blood preservatives have claimed206-208 or implied 1 2 5 . 1 9 7 - 2 0 1 that their

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